Ž .Biophysical Chemistry 79 1999 1]9

Paramagnetic fluorescence quenching in a modelmembrane: a consideration of lifetime and temperature

David A. JohnsonU, Binh Nguyen, Anibal F. Bohorquez,C. Fernando Valenzuela1

Di ision of Biomedical Sciences, Uni ersity of California, Ri erside, CA 92521-0121, USA

Received 22 December 1998; accepted 3 January 1999

Abstract

To expand our understanding of paramagnetic quenching in membranes, the relationship between fluorophoreŽ .excited-state lifetime t , temperature, and the collisional quenching was studied. Specifically, the ability of tempo to

Žquench the steady-state and time-resolved emission from five lipophilic fluorophores diphenylhexatriene, perylene,. Ž .phenanthrene, pyrene, and triphenylene partitioned into egg phosphatidylcholine EggPC liposomes was examined.

ŽAlso, the temperature dependence of spin-labeled androstane to quench the emission steady-state and time-.resolved from perylene in EggPC liposomes was determined. Unexpectedly, in EggPC liposomes, the apparent

quenching efficiency decreased with increasing t until the effect leveled off above ;20 ns. Moreover, in EggPCliposomes, dynamic quenching decreased with increasing temperature. The results suggest that in membranes,paramagnetic quenching is more complex than generally recognized. Q 1999 Elsevier Science B.V. All rightsreserved.

Keywords: Paramagnetic fluorescence quenching; Fluorescent probe; Spin label; Liposomes; Time-resolved fluorescence

1. Introduction

Molecular interactions in biological mem-branes have been increasingly studied by means

Abbre¨ iations: ASL, 5-doxyl-17b-hydroxy-5a-androstane;DPH, 1,6-diphenyl-1,3,5-hexatriene; EggPC, Egg phos-phatidylcholine; tempo, 2,2,6,6-tetramethylpiperdine-1-oxyl.U Corresponding author. Tel.: q1-909-787-3831; fax: q1-909-787-5504; e-mail: david.johnson@ucr.edu

1Present address: Department of Neuroscience, Universityof New Mexico HSC, Albuquerque, NM 87131

of fluorescence quenching with stable nitroxylŽ .free radicals spin-labels . Examples of the issues

pursued by paramagnetic quenching with spin-labels include: the determination of the affinity of

w xlipids toward membrane proteins 1 , the assess-ment of the transverse position of intrinsic and

w xextrinsic fluorophores 2]4 , the evaluation of lipidw xphase separation 5 , and the localization of lig-

w xand-binding sites on receptors 6]10 .It has been recognized for some time that no

Ž .matter what the excited-state lifetime t of thefluorophore is, spin-label-induced quenching in

0301-4622r99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S 0 3 0 1 - 4 6 2 2 9 9 0 0 0 3 6 - 8

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]92

non-viscous, isotropic solvents will display bothŽ .dynamic reduced fluorescence lifetime and static

Žloss of fluorescence without changes in fluores-.cence lifetime components. In lipid membranes,

on the other hand, the situation is more complex.Up until about a decade ago, it has been gener-ally assumed that spin-label-induced quenching inmembranes is only static for fluorophores withexcited-state lifetimes of less than ;20]50 ns.The reason for this assumption is that dynamic

Ž .quenching not involving dipolar energy transferis diffusion-dependent and that fluorophorerquencher translational diffusion in membranes is

Ž 7 8 2 .too slow 10 ]10 cm rs to produce a significantnumber of collisions within the duration of the

w xexcited state of the fluorophore 1 . However,w xMerkle et al. 11 demonstrated that spin-label-in-

duced dynamic quenching, in fact, occurs in mem-branes with short-lived fluorophores. Fast local

Žmotions i.e. wobbling, rotation, gauche-trans.isomerization, vertical fluctuations of the spin-

label andror the fluorophore are postulated tow xallow for this dynamic quenching 11,12 .

Short-lived lipophilic fluorophores should,therefore, have the capacity to monitor fast mo-tions of membrane-associated fluorophores andspin-labels and, thus, provide additional uses forparamagnetic quenching. While the experimentalsupport for this local-motion hypothesis is com-pelling, the phenomena have not been completelycharacterized. For example, it is unclear whetherthere is a unique relation between the t and thequenching. Also, the effect of temperature on thisquenching has not been examined.

Consequently, to expand our understanding ofparamagnetic quenching in membranes, we ex-amined in detail the relation between fluorophorelifetime and temperature on paramagneticquenching. Specifically, the differential ability ofa spin-label, tempo, to quench the emissionŽ .steady-state and time-resolved from five

Žlipophilic fluorophores diphenylhexatrieneŽ .DPH , perylene, phenanthrene, pyrene, and

.triphenylene that were partitioned into egg phos-Ž .phatidylcholine EggPC liposomes was examined.

Also, the temperature dependence of spin-labeledŽ .androstane ASL to quench the emission

Ž .steady-state and time-resolved of one of these

Ž .fluorophores perylene was determined to assesswhether or not dynamic quenching increased withtemperature as generally assumed. Two novelobservations about paramagnetic quenching in

Ž .model membranes were made: 1 the apparentbimolecular collision rates for the series of fluo-rophores decreased with increasing t until it lev-

Ž .eled off above ;20 ns, and 2 dynamic quench-ing decreases with increasing temperature. Theresults appear to suggest a greater complexity toparamagnetic quenching in membrane than isgenerally recognized.

2. Materials and methods

2.1. Materials

Triphenylene, perylene, phenanthrene, andŽ .2,2,6,6-tetramethylpiperdine-1-oxyl tempo wereŽobtained from Aldrich Chemical Co. Milwaukee,

. Ž .WI . 1,6-diphenyl-1,3,5-hexatriene DPH wasŽ .purchased from Molecular Probes Eugene, OR .

Pyrene was obtained from Sigma Chemical Co.Ž .St. Louis, MO . EggPC was purchased from Cal-

Ž .biochem La Jolla, CA. .

2.2. Preparation of EggPC liposomes

Multilamellar lipid dispersions were made bydissolving EggPC in a 1:1 methanolrchloroformmixture and rotary evaporating the mixture untildry. The sample was then placed under a vacuumŽ .-1 mtorr for at least 30 min. The thin film wasthen hydrated with warm 10 mM NaPO , pH 7.44Ž .Buffer I and vortexed for 5 min in the presenceof glass beads.

2.3. Fluorescence measurements

Steady-state fluorescence measurements weremade with a Perkin-Elmer Cetus MPF 66 spec-trofluorometer. Fluorescence lifetimes were de-termined by the time-correlated single-photoncounting technique using an EEY Scientific

Ž .nanosecond spectrofluorometer La Jolla, CA.equipped with a high pressure hydrogen arc lamp.All titration were carried out in 0.5=0.5-cm cu-vettes, under argon, and at room temperature

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]9 3

unless stated otherwise. Fluorescence values werecorrected for dilution as a result of added titrantand the intrinsic absorbance of the sample as

w xdescribed by Valenzuela et al. 6 . Fluorescencedecay data was analyzed with Globals Unlimited

Žcomputer program Laboratory of Fluorescence.Dynamics, Urbana, IL. .

For the lifetime measurements excitation andemission wavelengths were selected for the vari-ous fluorophores with the following filter combi-

Ž . Žnations excitation, emission : perylene Oriel 400nm broad-band interference, Corning 3]71 cut-

. Žon , DPH Oriel 340-nm interference, Oriel 450-. Žnm broad-band interference , triphenylene Oriel

.280-nm interference, Oriel 59459 cuton , phenan-Žthrene Oriel 290-nm interference, Oriel 59460

. Žcuton , and pyrene Oriel 340-nm interference,.Schott KV-389 cuton . Pyrene fluorescence was

always measured with the sample under a nitro-gen atmosphere to minimize oxygen quenching.

2.4. Fluorescence quenching by tempo in cyclohexane

All stock solutions of fluorophores and tempowere initially dissolved in cyclohexane except forDPH, which was dissolved in methanol. Fluo-rophores were added to argon-saturated cyclohex-ane to give a final concentration of 6 mM. TempoŽ .1 M was added to the cuvettes with a Hamiltonsyringe. Fluorescence lifetimes were measuredbefore and after the addition of tempo.

The results were initially fit to the Stern]Volmer equation

w x Ž .t rts1qK Q 1o Q

where t and t are the fluorescence lifetimes of0the fluorophores in the absence and presence of a

w xconcentration Q quencher, respectively. K , theQStern]Volmer quenching constant is related tothe apparent bimolecular collision rate constantŽ .k by the expressionq

Ž .k sK rt . 2q Q o

In cyclohexane, where the bimolecular collisionŽ .rate constant k can be estimated with theo

Smoluchowski equation, the quenching efficiency

Ž .g or the quenching probability per encountercan be calculated with the expression

Ž .gsk rk . 3q o

2.5. Paramagnetic quenching in EggPC liposomes

Ž .The fluorophores 6 mM were added to aŽ .suspension of EggPC liposomes 600 mM and

incubated at room temperature for at least 90min prior to the start of each titration. The ef-fects of scattering were subtracted by measuringthe apparent emission from samples that includedall the ingredients except the fluorophore. Cor-rections were also made for dilution and innerfilter effects of the spin label. Samples containingDPH were protected from light. The effects ofthe quencher upon both the steady-state andtime-resolved emission were measured and the

Ž .data were plotted Stern]Volmer plot as eithersteady-state intensity in the absence of quencherover the intensity in the presence of quencher ort rt vs. quencher concentration. The slopes of0

Ž SS .these plots are equal to the steady-state KQŽ .and time-resolved K Stern]Volmer quenchingQ

constants, respectively.

3. Results

3.1. Determination of intrinsic quenching efficiencyof fluorophores

To have a more detailed understanding of thequenching characteristics of the fluorophores un-der study, the quenching efficiency of tempo todynamically quench the emission of the fluo-rophores was determined in an isotropic medium,

Ž .cyclohexane. Cyclohexane was chosen because: iits hydrocarbon composition is similar to the

Ž .membrane core; ii all the fluorophores underŽ .study are soluble in it; and iii its isotropic char-

acter provides a directionally independentquenching, which presumably should yield a moreaccurate estimate of the relative quenching effi-ciencies of the fluorophores.

In cyclohexane the emission decays of DPH,perylene, and pyrene were well fit to a single

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]94

exponential equation, while those of phenan-threne and triphenylene were best fit to a biexpo-

Ž .nential equation data not shown . Because of thecomplexity of the phenanthrene and triphenyleneemission decays, the geometric average of the twolifetimes was utilized to calculate the bimolecularcollision rate constants. The time-resolved

Ž .Stern]Volmer plots for tempo to shorten quenchthe excited-state lifetimes of the fluorophores ex-

Ž .amined are shown in Fig. 1. The slopes K ofQlinear least-squares fits to these plots along with

Ž .the apparent collisional rate constants k andq

Table 1Summary of the isotropic Stern]Volmer quenching constants,decay rates, apparent collisional rate constants, and quenchingefficiencies for tempo quenching the emission from fivefluorophores in cyclohexane

a b y9c dFluorophore K t k =10 gQ 0 qy1 y1y1Ž . Ž . Ž .M ns M s

DPH 164 11.5 14.2 2.2Perylene 65 4.5 14.5 2.2

ePhenanthrene 194 42.2 4.6 0.7Pyrene 2023 180 11.2 1.7

eTriphenylene 266 37.9 7.0 1.1

a Ž Ž ..Stern]Volmer quenching constant calculated Eq. 1Žfrom the slopes of the data plots in Fig. 1 Each value

.represents the mean of 3]5 determinations .b Fluorescence lifetime in cyclohexane in the absence of

quencher.cApparent bimolecular quenching rate constant calculated

Ž .using Eq. 2 .dQuenching efficiency estimated by dividing k by k , theq 0

theoretical association rate for diffusion of the fluorophoreŽ .and the quencher toward each other calculated with Eq. 3 .

The values of k ranged between 6.54 and 6.57=109 My1o

sy1 .The theoretical k can be estimated with the Smolu-0chowski equation

Ž .Ž .Ž .k s 4p Nr1000 R qR D qDo f q f q

where N is the Avogadro’s number, and R and D are theŽ .radius and diffusion coefficients of the fluorophore f and

Ž .quencher q . D can be estimated using the Stokes]Einsteinequation

DskTr6phR

Ž .where k is the Boltzmann’s constant, T the temperature 8K ,and h is the viscosity of the media.

eGeometric average lifetime calculated with the expressionSa t rSa , where a and t are the preexponential term andi i i i ilifetime of the ith decay component, respectively.

Ž .the quenching efficiencies g are presented inTable 1. All the plots were linear and the appar-ent bimolecular collisional rate constants rangedbetween 0.46 and 1.5=1010 My1 sy1, consistentwith a simple diffusion-controlled quenchingprocess. The estimated quenching efficiencies, g ,which are the apparent collisional rate constantsdivided by the theoretical bimolecular association

Žrates determined with the Smoluchowski expres-.sion , ranged between 0.7 and 2.2 and were in the

rank order of DPH s perylene ) pyrene )triphenylene )phenanthrene. The greater thanunity value of these quenching efficiencies is con-sistent with the effective encounter distance ofthe spin label extending a few angstroms beyond

w xits van der Waals radius 13 . Also, the threefoldvariation in these quenching efficiencies showsthat tempo is not equally effective in quenchingall fluorophores even when the chemical composi-tions of the fluorophores are relatively similar.

Ž .And, not surprisingly, no correlation rsy0.1between t and g was observed, which shows thatthe excited-state lifetime of the fluorophores isunrelated to the efficacy of a spin label to quenchemission in cyclohexane.

3.2. Paramagnetic quenching in EggPC liposomes

The tempo-induced quenching of both thesteady-state and time-resolved emission of thevarious fluorophores under study were de-termined and the Stern]Volmer plots of the re-sults are illustrated in Fig. 2. The emission decaysof all the fluorophores were complex and best fitto a biexponential equation, Consequently, thegeometric averages of the decay parameters wereutilized to calculated the bimolecular collisionrate constants. Also, to make quantitative com-parisons, the total concentration of tempo addedat each step of the titrations was used to calculatethe apparent quenching constants even thoughonly a fraction of the tempo partitions into theliposomes.

Over the range of quencher concentrations ex-amined all the Stern]Volmer plots were reason-ably linear. For the fluorophores examined, thesteady-state Stern]Volmer quenching constants

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]9 5

Fig. 1. Stern]Volmer plots for tempo quenching the emission from the five fluorophores in cyclohexane. Quenching is measured asŽ .a reduction in the fluorescence lifetime and the various symbols B, `, and v represents the results of separate experiments. The

lines represent linear least-squares fits of the data.

were from 1.1- to 4.6-fold higher than the compa-Ž .rable time-resolved constants Table 2 .

Division of the time-resolved Stern]Volmerquenching constants by the respective lifetimes

Ž Ž ..Eq. 2 yields the apparent bimolecular collisio-nal rate constants, k ’s. The descending rankqorder of these constants is perylene)DPH)phenanthrene )pyrene) triphenylene, which is

Fig. 2. Stern]Volmer plots for tempo quenching the emission of the five fluorophores in EggPC liposomes. Quenching wasŽ . Ž .measures as either a reduction of the steady-state emission v or fluorescence lifetime ` of the same sample. The error bars

Ž .represent the S.D. of three determinations. The lines represent linear least-squares fits of the data. The fluorophores 6 mM wereŽ . Ž .added to multilamellar EggPC liposomes 600 mM in 10 mM sodium phosphate buffer pH 7.4 and incubated for 1]2 h at room

temperature. Corrections for dilution, intrinsic fluorescence of the sample, and absorbance of the quencher were made in all cases.

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]96

Table 2Summary of the steady-state and time-resolved Stern]Volmer quenching constants, apparent collisional rate constants for tempoquenching of the emission from five fluorophores in EggPC liposomes

cSS a b y9 d y9 e² :K K t k =10 k rg=10Q Q q qy1y1 y1 y1Ž . Ž . Ž . Ž .M M ns M s

DPH 500 240 7.5 32.3 14.9Perylene 240 150 2.9 52.1 23.7Phenanthrene 1600 350 19.7 17.6 25.0Pyrene 1100 980 105.9 9.3 5.4Triphenylene 410 180 28.1 6.3 5.9

aApparent Stern]Volmer quenching constant calculated from steady-state quenching data illustrated in Fig. 2.b Ž Ž ..Apparent Stern]Volmer quenching constant calculated Eq. 1 from the slopes of the lifetime quenching data illustrated in

Fig. 2.cGeometric average lifetime calculated with the expression Sa t rSa , where a and t are the preexponential term and lifetimei i i i i

of the ith decay component, respectively.d Ž .Apparent bimolecular collision rate constant using Eq. 2 and K .Qe Ž . Ž .Apparent bimolecular collision rate constant k divided by the quenching efficiency g from Table 1.q

different from the comparable rank orderobserved with the fluorophores dissolved in cy-

Žclohexane perylene s DPH ) pyrene ).triphenylene ) phenanthrene . The differential

ability of tempo to quench the emission fromfluorophores in cyclohexane vs. the hydrocarboncore of EggPC liposomes is quantitatively seen bydividing the EggPC k ’s by the quenching effi-q

Ž .ciencies, g , determined in cyclohexane Table 2 .If tempo quenched equaled the emission fromfluorophores in both cyclohexane and EggPCliposomes, then the k rg ratios would be aboutqthe same, which they are not. These ratios differfrom one another by as much as a 4.6-fold. Whythese differences occur is unclear but it mayreflect differences in the membrane distributionof the fluorophores andror orientation-depen-dent differences in their quenching efficiencies.

The relation between the apparent bimolecularcollisional rate constant and t is different incyclohexane and EggPC liposomes. In cyclohex-ane, as discussed above, no systematic relationwas observed; however, in EggPC liposomes theapparent bimolecular collisional rate constantsand, therefore, the relative quenching efficiencydecreases with increasing lifetime until the effect

Ž .levels off above a t of ;20 ns Fig. 3 . Essen-tially, this same relationship is seen if the steady-state instead of the time-resolved Stern]Volmer

Ž .quenching constants are used data not shown ;

Fig. 3. Relation between the fluorescence lifetime of thefluorophores and their apparent bimolecular rate constants

Ž .for tempo quenching in EggPC liposomes v and in cyclohex-Ž .ane ` .

consequently, this phenomena is unrelated to thestatic quenching elements in spin-labeled-inducedloss of fluorescence.

3.3. Temperature dependence of quenching

To further characterize spin label fluorescencequenching in membranes, the effect of tempera-ture on ASL quenching of the emission fromperylene in EggPC liposomes was examined. ASL

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]9 7

was utilized instead of tempo, because the mem-brane partitioning of tempo can change with tem-perature. ASL, a steroid, on the other hand,rapidly and essentially completely partitions intomembranes at room temperature, and, therefore,its membrane concentration should not vary over

Ž .the relatively small temperature range 158Cstudied.

The temperature dependence of ASL-inducedquenching of both the steady-state and time-re-solved emission of perylene was determined, andthe results are illustrated in Fig. 4. One set of the

Ž .Stern]Volmer plots at 208C is shown as anexample in the upper panel of Fig. 4. As with thecomparable tempo results, the Stern]Volmerplots were essentially linear over the concentra-tion range of ASL examined, and the steady-statedata were associated with greater degree ofquenching than those obtained from the time-re-solved results. Because ASL essentially com-pletely partitions while tempo partially partitionsinto membranes, the apparent Stern]Volmerquenching constants associated with ASL-inducedquenching were ;50-fold greater than those in-duced by tempo. Significantly, the ratios of thesteady-state and time-resolved Stern]Volmerquenching constants were about the same for

Ž . Ž .both spin labels ASL and tempo , 1.8 at 208CŽ .vs. 1.6 at ;228C , respectively, indicating that

once partitioned into the membrane the two spinlabels were equally effective quenchers.

Surprisingly, while there was little or no effectof increasing temperature on the steady-stateStern]Volmer quenching constant, the time-re-solved Stern]Volmer quenching constant de-

Žcreased with increasing temperature Fig. 4, lower. Žpanel . From 15 to 308C, the dynamic time-re-.solved quenching constant decreased by approxi-

mately 36%. This effect cannot be explained by atemperature-dependent increase in the fluores-cence lifetime of perylene, because the lifetime of

Ž .perylene only decreased slightly 4% as the tem-Žperature was increased from 15 to 308C data not

.shown . While it is unclear why this effect occurs,the results are inconsistent with the typical behav-ior of dynamic quenchers in isotropic environ-ments, where the frequency of collisions and

w xquenching increase with temperature 14 .

Fig. 4. Temperature dependence of the steady-state and thetime-resolved quenching of perylene by ASL in EggPC lipo-somes. Upper Panel: Stern]Volmer plots of ASL quenching

Ž .either the steady-state emission v or the fluorescence lifetimeŽ . Ž .` from perylene 6 mM partitioned into EggPC liposomesŽ .600 mM at 208C. The structure of ASL is shown as an inset.

Ž .Lower panel: The apparent steady-state v and the time-re-Ž .solved ` Stern]Volmer quenching constants as a function

of temperature for the quenching of perylene emission byASL in EggPC liposomes. The error bars represent the S.D. offive to six determinations, and the lines represent linearleast-squares fits of the data.

4. Discussion

Fluorescence quenching can be dynamicandror static. Quenching that competes withemission to accelerate the excited-state-to-ground-state relaxation is dynamic and involveseither a collision with or resonance energy trans-fer to a quencher. Quenching that occurs withouta change in the fluorescence lifetime of the fluo-rophore typically involves ground-state pheno-mena where the quencher either forms a darkcomplex that absorbs, but does not emit, or aground-state complex that displays a reduced ex-tinction coefficient relative to the free fluo-rophore. In non-viscous, isotropic environments

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]98

paramagnetic quenchers reduce emission by bothdynamic and static mechanisms. Both types ofquenching occur with paramagnetic quenchers,because the effective radii of these quenchers

˚extend 4]6 A beyond their van der Waals radiiw x13 , which effectively prolongs the dwell time ofthe collisional encounter to produce instanta-neous de-excitation and a dark complex.

In this paper, we set out to expand our under-standing of paramagnetic quenching by spin labelsin membranes. The relationships between t , tem-perature, and paramagnetic quenching in EggPCliposomes were explored. Two unexpected obser-vations were made: first, in model membranes,unlike in isotropic media, fluorophores withlifetimes below ;20 ns were more effectivelyquenched by spin labels than those with longert ’s. Second, dynamic quenching decreases withincreasing temperature, contrary to the typicallyobserved increase with temperature in isotropicsolvents.

The lipid bilayer is an anisotropic mediumwhere lipid movements are much slower than innon-viscous media and are largely constrained totwo dimensions. Fluorophores with short excited-state lifetimes should be associated with a shorterperiod during which an encounter can occur withquenchers, which will proportionally reducequenching from spin labels at greater distances.The routine division of the observed Stern]Volmer quenching constants by t to calculate theapparent bimolecular collision rate constant, inessence, compensates for the inherent property ofexcited-state lifetime to influence the apparentquenching efficiency. Indeed, this compensationeliminates the apparent lifetime dependence of

Ž .quenching in cyclohexane Fig. 3 , but not inEggPC liposomes. The reason for this pheno-menon is unclear. It may some how involve theinherent heterogeneity in the bilayer microenvi-ronment of each fluorophore and thus multiplic-ity of excited states, each of which with differen-tial sensitivity to paramagnetic quenchers.

The reason for the temperature-dependent de-crease in the time-resolved but not steady-statequenching of perylene by ASL in EggPC lipo-somes is also unclear. For there to be a change in

the time-resolved quenching without a change inthe steady-state quenching, the dynamic and staticcomponents of the quenching must be recipro-cally linked, such that as dynamic quenching de-creases with increasing temperature staticquenching must increase. Because time-resolvedemission is only sensitive to dynamic quenchingand steady-measurements represent the sum ofboth static and dynamic quenching, a reciprocalshift from dynamic to static quenching would beobserved as a decreasing time-resolved quenchingand unchanging steady-state quenching.

This reciprocal shift on the level of dynamic vs.static quenching of perylene emission by ASLcould be due to a temperature-dependent redis-tribution of ASL in the bilayer. This could occur,for example, if the non-polar, disc-shaped pery-lene distributed primarily between the two bilayerleaflets, parallel to the plane of the bilayer, andthe more polar ASL distributed primarily at lowertemperatures between phosphatidylcholinemolecules, perpendicular to the plane of the bi-layer. If elevation of temperature shifted the dis-tribution of ASL toward the middle of the bilayer,then the perylene would increasingly be withinthe effective encounter distance of ASL and morelikely be instantaneously and, therefore, staticallyquenched.

5. Conclusion

In summary, we found that the quenching ef-ficiency of spin labels has some dependence upon

Ž .t t-20 ns when fluorophores are partitionedinto lipid bilayers, but not when dissolved in anon-viscous, isotropic medium like cyclohexane.The reason for this phenomenon is unclear, butmay reflect a differential sensitivity of the fluo-rophores to paramagnetic quenching in the uniquemicroenvironment of a lipid membrane. Additio-nally, we found that temperature can shift theproportion of dynamic-to-static quenching ofperylene emission by ASL. This phenomenon mayresult from a temperature-dependent redistribu-tion of the ASL from between lipid moleculesperpendicular to the plane of the bilayer to thespace between the two bilayer leaflets.

( )D.A. Johnson et al. r Biophysical Chemistry 79 1999 1]9 9

Acknowledgements

This work was supported by NSF grant IBN-9515330.

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