Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence...

Determination of the depth of BODIPY probes in model membranes byparallax analysis of £uorescence quenching

Robert D. Kaiser a, Erwin London a;b;*a Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA

b Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA

Received 3 February 1998; accepted 21 May 1998

Abstract

The location of a series of lipophilic and lipid-attached BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) membraneprobes was analyzed by the quenching of BODIPY fluorescence by a series of nitroxide-labeled lipids in which the depth ofthe nitroxide group is varied. When attached to the polar headgroup of PE the BODIPY remained near the polar headgroupin depth. However, when attached at the end of free or phospholipid-attached fatty acyl chains, or when attached to twohydrocarbon chains, we observed two probe populations. One, usually dominant, population of BODIPY groups `loopedback' towards the surface, but a second population remained deeply embedded within the bilayer. When attached to a fattyacid or fatty acyl chain, the deep population appeared to locate at a depth related to its point of attachment to the acyl chain.In BODIPY linked to free fatty acids, the location of the deep population responded to the ionization of the carboxyl group.Because, unlike NBD (7-nitro-2,1,3-benzoxadiazol-4-yl) and most dansyl groups, acyl chain linked BODIPY groups can existin a deeply buried form we conclude that BODIPY linked acyl chains are superior to NBD or dansyl linked acyl chains asmembrane probes. ß 1998 Elsevier Science B.V. All rights reserved.

Keywords: Liposome; Cholesterol ; Membrane probe; Fluophore distribution; Spin-label

0005-2736 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 3 6 ( 9 8 ) 0 0 1 2 7 - 8

Abbreviations: BODIPY, 4,4-di£uoro-4-bora-3a,4a,diaza-s-indacene; BODIPY-PE, N-(4,4-di£uoro-5,7-dimethyl-4-bora-3a,4a,diaza-s-indacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; C1-BODIPY-C12-COOH, 4,4-di£uoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; C8-BODIPY-C5-COOH, 4,4-di£uoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid;C10-BODIPY-C3-COOH, 5-decyl-4,4-di£uoro-4-bora-3a,4a-diaza-s-indacene-3-propionic acid; C1-BODIPY-C12-PC, 1-hexadecanoyl-2-(4,4-di£uoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-sn-glycero-3-phosphocholine C8-BODIPY-C5-PC, 1-hexadecanoyl-2-(4,4-di£uoro-5-octyl-4-bora-3a,4a,diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine; didecyl-BODIPY, 3,5-didecyl-4,4-di£uoro-4-bora-3a,4a,diaza-s-indacene; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; ESR, electron spin resonance; methyl4-BODIPY, 4,4-di£uoro-1,3,5,7-tetramethyl-4-bora-3a,4a,diaza-s-indacene; MLV, multilamellar vesicles; NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl ; OG, n-octyl-L-D-glucopyranoside (octyl glucoside); PC, 1,2 diacyl-sn-glycero-3-phosphocholine; 5-SLPC or 12-SLPC, 1-palmitoyl-2-(5- or 12-doxyl) stearoyl-sn-glycero-3-phosphocholine; SUV, small unilamellar vesicles; Tempocholine: 4-(N,N-dimethyl-N-(2-hydroxyethyl)) am-monium-2,2,6,6- tetramethyl piperidine-1-oxyl; TempoPC, 1,2-dioleoyl-sn-glycero-3-phosphotempocholine; TLC, thin-layer chromatog-raphy

* Corresponding author. Fax: +1 (516) 632-8574; E-mail : [email protected]

BBAMEM 77445 24-9-98

Biochimica et Biophysica Acta 1375 (1998) 13^22

1. Introduction

One of the fundamental questions involved in thestudy of membrane structure and function is: whatchemical and structural factors control the structureand depth of molecules in membranes? Fluorescentprobes have proven a powerful approach to obtainanswers to these questions. However, the interpreta-tion of £uorescent probe behavior depends inlarge part upon where it is located in the bilayer,and for many probes this has been only poorlycharacterized. In fact, in more than one case, £uo-rescent probes have been found to localize in a re-gion of the membrane other than that for which theywere originally designed [1^3]. In most other cases,only a rough estimate of probe location has beenavailable.

To help locate molecules in membranes we devel-oped a £uorescence quenching technique called par-allax analysis, which allows the depth of a £uores-cent group in a lipid vesicle to be determined fromthe result of two quenching experiments [1,3,4]. Inone experiment, the £uorescence intensity of a £uo-rophore in the presence of a nitroxide (spin)-labeledquencher located at a speci¢c depth is measured. In asecond experiment £uorescence is measured in thepresence of a quencher carrying the nitroxide groupat a di¡erent depth. The ratio of the two intensities isthen substituted into a equation that allows calcula-tion of depth at a high level of resolution. Parallaxanalysis has been applied to a large number of di¡er-ent £uorescent probes, polypeptides and proteins,and several tests con¢rming its accuracy have beenmade [1,3^11].

In this report, the location of BODIPY probes inmembranes is examined. BODIPY probes are a re-cently introduced class of £uorescent probes thathave excellent £uorescent properties. They have ahigh extinction coe¤cient, £uorescence quantumyield, and photostability [12,13]. They £uoresce inthe visible, with excitation and emission spectra sim-ilar to those of the widely used xanthene dyes (£uo-rescein, rhodamine, eosin, Texas red, etc.) [12,13].However, unlike xanthene dyes the BODIPY groupcarries no net charge and therefore can be used as aprobe of membrane structure. Furthermore, it is be-lieved that BODIPY groups are signi¢cantly morehydrophobic than previously used polar membrane

probes such as NBD. As a result, BODIPY probeshave been used in a number of studies of the struc-ture and function of membranes and individual lipids[14^19]. In this study, we ¢nd BODIPY groups havea tendency to move toward the polar headgroup re-gion of the bilayer. However, a considerable popula-tion of BODIPY molecules can remain buried withinthe lipid bilayer. Therefore, BODIPY appears to be auseful £uorescent analog for studies of lipid behav-ior.

2. Materials and methods

2.1. Materials

BODIPY probes were purchased MolecularProbes (Eugene, OR). The purity of the BODIPYprobes was checked by TLC on silica gel plates (Sili-ca gel H, Uniplate, Newark, DE) using solvent sys-tems of: 10/30 CHCl3/methanol (v/v) for C10-BOD-IPY-C3-COOH, C1-BODIPY-C12-COOH, and C8-BODIPY-C5-COOH; 32/25/2 CHCl3/Methanol/H20(v/v) for C1-BODIPY-C12-PC, C8-BODIPY-C5-PC, and BODIPY-PE; 30/10 mixed hexanes/CHCl3(v/v) for didecyl-BODIPY; 20/20 mixed hexanes/CHCl3 (v/v) methyl4-BODIPY. All probes gave asingle intensely colored spot.

2.2. Assay of spin-label content of spin-labeled lipids

Nitroxide-labeled PCs and DOPC were purchasedfrom Avanti Polar Lipids (Pelham, AL). The purityof phospholipids was con¢rmed by TLC on silica gelplates as described previously [3]. The concentrationof phospholipids was determined by phosphate assaysubsequent to total digestion [3]. The actual nitroxidecontent of the nitroxide-labeled lipids was calculatedfrom the intensities of the doubly integrated ESRspectra as described previously [1]. Alternatively,the concentration of the spin-labeled lipids was as-sayed with £uorescence quenching by determiningthe % of nitroxide-labeled lipid that had to be incor-porated into vesicles to give the same quenching ofanthroyloxy fatty acids as that found previously forvesicles containing 15% nitroxide-labeled lipids [3].The ratio of nitroxide groups to lipid was generallyfound to be in the range 0.6^0.9.

BBAMEM 77445 24-9-98

R.D. Kaiser, E. London / Biochimica et Biophysica Acta 1375 (1998) 13^2214

2.3. Preparation of samples for £uorescencemeasurements

Depth measurements were made on samples con-taining BODIPY derivatives incorporated into200 WM SUVs prepared by octylglucoside (OG) di-lution [20]. Solutions of 2.5 WM BODIPY derivativedissolved in 25 mM OG were prepared by dryingaliquots of OG and BODIPY derivatives dissolvedin ethanol with N2 and then dissolving in H2O. In

separate tubes ethanol solutions containing 1 Wmolof OG and 200 nmol of total phospholipid contain-ing DOPC, or DOPC with 15 mol% 5-SLPC, 12-SLPC or TempoPC1 were mixed and then driedwith N2. Then 40 Wl of the BODIPY derivative in

Fig. 1. Structure of the BODIPY probes studied in this report.

1 The nitroxide-labeled lipid contains some molecules that lacka nitroxide group. The amount of nitroxide-labeled lipid is ad-justed to give 15% nitroxide-containing molecules and 85% otherlipid molecules (DOPC plus inactive `quencher').

BBAMEM 77445 24-9-98

R.D. Kaiser, E. London / Biochimica et Biophysica Acta 1375 (1998) 13^22 15

OG was added and the samples vortexed to dissolveall the components. Lastly, 960 Wl of bu¡er (10 mMNa acetate/150 mM NaCl, pH 4.5; 10 mM Na phos-phate/150 mM NaCl, pH 7 (PBS); or 10 mM glycine/150 mM NaCl, pH 10) was added to each tube andthe tubes were vortexed for 30 s. In samples contain-

ing cholesterol, an additional 100 nmol cholesterolwas included with the phospholipid. Final BODIPYconcentration was 0.1 WM.

2.4. Fluorescence quenching measurements

Fluorescence was measured in 1 cm path lengthquartz cuvettes using a Spex Fluorolog spectro-£uorimeter operating in ratio mode. The excitationand emission slits were set at 1.25 mm (2.3 nm band-pass) to prevent overloading of the photomultipliertube. The excitation wavelength was set at 500 nm,and emission at 530 nm. Control experiments showedthat £uorescence intensities were stable over severalminutes, and that preincubation in the dark or inroom light did not a¡ect intensity. The £uorescenceintensity from duplicate or triplicate samples con-taining £uorophore was averaged. The intensity ofthe background samples without £uorophore wasfound to be negligible (6 1%). All measurementswere made at room temperature. The ratio of the£uorescence intensities in the presence (F) and inthe absence (Fo) of the nitroxide-labeled lipids (F/Fo) was calculated and substituted into the parallaxequation [1,3] to calculate BODIPY depth (see be-low).

To con¢rm the absence of reactivity between nitro-xide-labeled lipids and BODIPY probes, in controlexperiments £uorescence was measured after 100 Wlaliquots of each vesicle sample were dissolved in900 Wl ethanol. This abolished quenching and equal£uorescence intensities were observed in sampleswith and without nitroxide-labeled lipids.

Fig. 2. Binding of BODIPY probes to model membranevesicles. F/Fo is the ratio of £uorescence in the presence ofDOPC vesicles containing 15 mol% 5-SLPC to that in 100%DOPC vesicles. Binding is shown for: didecyl-BODIPY (P) atpH 7; (+) methyl4-BODIPY at pH 7; and (R) C1-BODIPY-C12-COOH, (a) C8-BODIPY-C5-COOH, and (b) C10-BODI-PY-C3-COOH at pH 10.

Table 1Nitroxide quenching and depth of BODIPY derivatives in DOPC at pH 7.0

Fluorophores Ftc F5 F12 zcf

Methyl4-BODIPY 0.53 þ 0.03 0.58 þ 0.02 0.57 þ 0.03 18.4 þ 0.8a

Didecyl-Bodipy 0.20 þ 0.03 0.35 þ 0.05 0.32 þ 0.03 21.4 þ 1.1BODIPY-PE 0.27 þ 0.05 0.41 þ 0.04 0.43 þ 0.01 20.3 þ 0.9C8-BODIPY-C5-PC 0.26 þ 0.04 0.38 þ 0.02 0.40 þ 0.02 19.7 þ 0.8C1-BODIPY-C12-PC 0.28 þ 0.01 0.40 þ 0.01 0.35 þ 0.01 19.4 þ 0.4

(7.2 þ 0.4)b

Ftc, F5 and F12 equal ratio of £uorescence intensity in the presence of the TempoPC, 5-SLPC or 12-SLPC, respectively, to that in theabsence of quencher (Fo). Values shown are for the average of 4-8 samples þ S.D.aThis value was calculated after correction of £uorescence quenching values for probe (about 33%) not bound to membranes [6].bThe value in parentheses is the upper limit to zcf of the deep subpopulation of probes calculated from the ratio of £uorescence by 5-SLPC and 12-SLPC. See Section 2 for details.

BBAMEM 77445 24-9-98


2.5. Calculation of depth by parallax analysis

Using F/Fo values the distance of £uorophoresfrom the center of the bilayer was calculated usingthe parallax equation [1,3]:

zcf � Lc1 � �3ln�F1=F2�=ZC3L221�=2L21;

where zcf is the distance of the £uorophore from thecenter of the bilayer, F1 is the £uorescence intensity(F/Fo) in the presence of the shallow quencher(quencher 1), F2 is the £uorescence intensity (F/Fo)in the presence of the deeper quencher (quencher 2),Lc1 is the distance of the shallow quencher from thecenter of the bilayer, L21 is the distance between theshallow and deep quenchers, and the C is the con-centration of quencher in molecules/Aî 2=(mole frac-tion of nitroxide-labeled phospholipid/area per phos-pholipid)=(mole fraction nitroxide/70 Aî 2) [1]. Thequenching by the pair of quenchers (i.e. nitroxide-labeled lipid) that quench the most (i.e. the Tem-poPC/5-SLPC pair or 5-SLPC/12-SLPC pair) wasused to calculate zcf [3]. The values used for the dis-tances of the nitroxide group from the bilayer center

were 5.85 Aî for 12-SLPC, 12.15 Aî for 5-SLPC, and19.5 Aî for TempoPC [1,3].

In model membranes containing cholesterol, the(lateral) concentration of nitroxide-labeled lipid andthe depth of the quenchers in the membranes arealtered. These values were calculated by assumingthat 33 mol% cholesterol results in a 10% increasein the width of the acyl chain region of the bilayer,and that a cholesterol molecule occupies a lateralarea of 32 Aî 2 [21,22]. The resulting value for C is(mole fraction of nitroxide-labeled phospholipid rel-ative to total lipid including cholesterol)/[0.67(70Aî )+0.33(32 Aî )]=(mole fraction nitroxide/57.5 Aî 2),and the values for the distances of 12-SLPC, 5-SLPC, and TempoPC nitroxides from the bilayercenter are, 6.45, 13.4 and 21 Aî , respectively2.

Table 2Nitroxide quenching and depth of BODIPY derivatives in DOPC/cholesterol at pH 7.0


Methyl4-BODIPY 0.59 þ 0.02 0.66 þ 0.01 0.64 þ 0.01 20.1 þ 0.6a

Didecyl-BODIPY 0.32 þ 0.02 0.44 þ 0.03 0.41 þ 0.01 20.7 þ 0.8BODIPY-PE 0.30 þ 0.01 0.43 þ 0.0 0.51 þ 0.003 21.4 þ 0.3C8-BODIPY-C5-PC 0.30 þ 0.01 0.42 þ 0.003 0.48 þ 0.02 20.8 þ 0.3C1-BODIPY-C12-PC 0.35 þ 0.05 0.48 þ 0.05 0.36 þ 0.03 20.7 þ 0.5

(7.0 þ 0.5)b

aThis value was calculated after correction of £uorescence quenching values for probe (about 33%) not bound to membranes [6].bThe value in parentheses is the upper limit to zcf of the deep subpopulation of probes calculated from the ratio of £uorescence by 5-SLPC and 12-SLPC. See Section 2 for details.

Table 3Nitroxide quenching and depth of BODIPY derivatives of free fatty acids in DOPC

Fluorophores Ftc F5 F12 ]zcf

C1-BODIPY-C12-COOH pH 4.5 0.23 þ 0.02 0.39 þ 0.02 0.33 þ 0.04 20.6 þ 0.6(7.1 þ 1.6)

C1-BODIPY-C12-COOH pH 10.0 0.32 þ 0.02 0.41 þ 0.01 0.43 þ 0.04 18.4 þ 0.8C8-BODIPY-C5-COOH pH 4.5 0.26 þ 0.05 0.37 þ 0.04 0.38 þ 0.01 19.5 þ 1.1C8-BODIPY-C5-COOH pH 10.0 0.29 þ 0.02 0.39 þ 0.01 0.44 þ 0.06 18.9 þ 1.1C10-BODIPY-C3-COOH pH 4.5 0.25 þ 0.05 0.38 þ 0.02 0.39 þ 0.01 20.4 þ 1.3C10-BODIPY-C3-COOH pH 10.0 0.24 þ 0.04 0.36 þ 0.02 0.41 þ 0.01 20.0 þ 1.3

2 There is very little di¡erence when the corrections forquencher depth and for concentration in the presence of choles-terol (see Section 2) are omitted. Therefore, the results are notsensitive to these corrections.

BBAMEM 77445 24-9-98


2.6. Upper limit of the depth of minor deepsubpopulations

Consider the case of a £uorophore that has dis-tinct shallow and deep subpopulations. In such asituation there are three di¡erent parameters thatcan be used to describe depth: the average depth ofall £uorophores zcf average; the average depth of thedeep £uorophore subpopulation, zcf deep; and theaverage depth of the shallow £uorophore population,zcf shallow. We are concerned with the case wherethe shallow population is near the headgroup and thedeep one much closer to the center of the bilayer.Where the major subpopulation is shallow, as ob-served for BODIPY probes, the depth calculatedfrom the parallax equation using the TempoPC and5-SLPC quenching will be close to zcf average [20],and is reported as such. In contrast, the depth calcu-lated from the parallax equation using the 5-SLPCand 12-SLPC quenching will be an upper limit to thedepth of the minor deep population (zcf deep) [20],and is reported as such in those cases where an ap-preciable deep population was detected. A more de-tailed discussion is given in reference [20].

2.7. Binding curves

Two sets of OG dilution SUVs with increasingamounts of lipid were prepared. Samples in bothsets ranged from 0 to 400 WM lipid. The ¢rst setcontained duplicate samples of DOPC and the sec-ond set contained duplicate samples of 15 mol% 5-SLPC mixed with DOPC. Both sets contained 0.1 WMBODIPY. To prepare these samples the appropriateamount of lipids was dried together with 1 Wmol of

DG and then the 40 Wl of BODIPY/OG solution wasadded and diluted as described above.

2.8. Fluorescence vs. pH; pKa

A bu¡er of 10 mM Na acetate/150 mM NaCl, pH5.0, was titrated to pH 3 with a small amount of 1 MHCl. Three sets of l.7 ml OG dilution SUV samplescontaining 200 WM lipid and 0.1 WM C8-BODIPY-C5-COOH were prepared in this bu¡er as describedabove. One set contained DOPC while the other setscontained 15 mol% 5-SLPC or 12-SLPC mixed withDOPC. The samples were placed in a 1 cm quartzcuvette, and both £uorescence and pH was meas-ured. Successive aliquots of base (NH4OH) wereadded to increase pH. Fluorescence was measured1^2 min later. The ¢nal £uorescence values were cor-rected for dilution.

3. Results

3.1. Binding of BODIPY probes to model membranevesicles

We applied £uorescence quenching to determinethe membrane location of a series of BODIPYprobes (Fig. 1) incorporated into model membranevesicles. The binding of BODIPY probes was ¢rstexamined in order to determine the best conditionsfor the quenching experiments. This was done bymeasuring the quenching detected when the probesbound to vesicles which contained a nitroxide-labeledlipid (Fig. 2). With the exception of the free methyl4-BODIPY probe which appears to bind half-maxi-

Table 4Nitroxide quenching and depth of free fatty acid BODIPY derivatives in DOPC/cholesterol


C1-BODIPY-C12-COOH pH 4.5 0.31 þ 0.01 0.45 þ 0.01 0.37 þ 0.04 21.2 þ 0.3(7.4 þ 1.1)

C1-BODIPY-C12-COOH pH 10.0 0.35 þ 0.03 0.47 þ 0.03 0.46 þ 0.05 20.4 þ 0.5C8-BODIPY-C5-COOH pH 4.5 0.33 þ 0.01 0.45 þ 0.02 0.44 þ 0.02 20.6 þ 0.3C8-BODIPY-C5-COOH pH 10.0 0.35 þ 0.01 0.45 þ 0.004 0.51 þ 0.02 19.8 þ 0.3C10-BODIPY-C3-COOH pH 4.5 0.31 þ 0.01 0.43 þ 0.03 0.46 þ 0.03 20.8 þ 0.5C10-BODIPY-C3-COOH pH 10.0 0.30 þ 0.004 0.43 þ 0.02 0.52 þ 0.01 21.1 þ 0.5

BBAMEM 77445 24-9-98


mally near 100^150 WM, the BODIPY probes wereall fully bound at 20 WM lipid3.

3.2. Measuring depth by £uorescence quenching

To measure the depth of the BODIPY probes their£uorescence was measured in unilamellar vesicles4

containing DOPC or DOPC mixed with 15% of shal-low (TempoPC), medium (5-SLPC), or deep (12-SLPC) nitroxide-labeled lipids (Tables 1^4). Strong£uorescence quenching was observed for all BODI-PY derivatives. The maximum amount of quenchingobtained (70^75%) was about the same for all but themethyl4-BODIPY probe, which was only partlybound to the vesicles under the conditions of theseexperiments. The amount of quenching obtainedwith the di¡erent nitroxide-labeled lipids was thenused to calculate the distance of the £uorophoresfrom the center of the bilayer (zcf ) using the parallaxequation (see Section 2. In the case of the methyl4-BODIPY probe this was done after correction forthe amount of £uorescence arising for unbound mol-ecules [6]).

3.3. Behavior of free and decyl linked BODIPYprobes

As shown in Tables 1 and 2 the methyl4-BODIPYprobe has a predominately shallow location withinthe polar headgroup region of the bilayer (zcf=18^20 Aî ). This can be seen qualitatively by the fact thatits is quenched most strongly by the shallow Tem-poPC probe (Tables 1 and 2). However, the quench-ing by the medium depth 5-SLPC and deep 12-SLPCis very similar, which is consistent with a minor sub-population of deeper BODIPY molecules or a verybroad distribution of BODIPY depths.

Didecyl-BODIPY also is quenched most stronglyby TempoPC and has a predominantly shallow pop-ulation (zcf=21 Aî ). Presumably this molecule is ori-ented with its BODIPY group near the membranesurface and its acyl chains penetrating the bilayeraligned with the lipid fatty acyl chains. There isalso a subpopulation of didecyl-BODIPY moleculeswith deep BODIPY groups, as shown by the slightlystronger quenching by 12-SLPC than 5-SLPC5.

3.4. Behavior of BODIPY attached to phospholipids

A more localized BODIPY depth is seen for BOD-IPY-PE, in which the BODIPY group is attached tothe polar headgroup amino group of PE. In this case,the BODIPY probe clearly has a shallow location(zcf=20^21 Aî ), and the fact that quenching by 12-SLPC is signi¢cantly weaker than that of the 5-SLPC suggests there is little if any deep subpopula-tion (Tables 1 and 2).

The behavior of BODIPY-PE is not surprisingsince the £uorescent moiety is attached to the head-group of a PE, designed so that it would be a veryshallow £uorophore. However, a similar predomi-nantly shallow location is observed for BODIPY de-rivatives of PC in which the BODIPY group is at-tached to one of the lipid acyl chains at a deep (C1-BODIPY-C12-PC) or intermediate position (C8-BODIPY-C5-PC) along the fatty acyl chain(zcf=19^21 Aî ). This implies that the BODIPY grouphas a su¤cient a¤nity for the polar region of themembrane to induce the fatty acyl chain to loop uptowards the surface, as has previously been seen forpolar derivatives of fatty acids [1^3, 20].

However, at least in the case of C1-BODIPY-C12-PC there is also a large population of BODIPYgroups that is deeply buried. This is shown by thestronger quenching by 12-SLPC than 5-SLPC. Thefact that quenching by the 12-SLPC is almost asstrong as that by TempoPC suggests the deep pop-ulation is a very considerable fraction of the total,and an upper limit to the depth of this population of

3 In most cases, there was a large increase in intensity in thepresence of lipid compared to that in its absence. Since BODIPY£uorescence is ordinarily insensitive to polarity [13], we assumethis re£ected an aggregation of the probes in solution in theabsence of lipid. Aggregation would result in BODIPY self-quenching, perhaps due to excimer formation [14].

4 We have repeatedly found the depth of small £uorescentprobes is not a¡ected by the size or curvature of the model mem-brane vesicles used [1,5,6].

5 It should be noted that it is di¤cult to pinpoint the depth ofminor subpopulations using parallax analysis [20], and generallywe have not attempted to do so. On the other hand when asecond subpopulation is large, a useful limit to zcf can be calcu-lated (see [20] and Section 2).

BBAMEM 77445 24-9-98


zcf =7 Aî (Tables 1 and 2) can be calculated from thequenching of the 5-SLPC and 12-SLPC (see Section2).

3.5. Behavior of BODIPY probes linked to free fattyacids: e¡ect of fatty acyl ionization on depth

The behavior of free fatty acid BODIPY deriva-tives is similar to that of PC with BODIPY fatty acylchains in many ways. The main population of BOD-IPY groups is located within the polar headgroupregion for all three BODIPY fatty acids (zcf=18^21Aî ). This is true whether the BODIPY is attachedclose to (C10-BODIPY-C3-COOH), or far from(C1-BODIPY-C12-COOH) the COOH group.

In addition, there is clearly a deep subpopulationof BODIPY groups for C1-BODIPY-C12-COOH atlow pH, similar to that noted above for the corre-sponding PC derivative. A distinct deep population,characterized by stronger 12-SLPC than 5-SLPCquenching, cannot be detected for the other BODI-PY fatty acids. This could re£ect either the lack ofsuch a population, or alternately that the deep pop-ulation is too close to the main population to beclearly discerned. In fact, the pH dependence of

quenching suggests that a deeper subpopulationdoes exist for these BODIPY fatty acids as well. Asin the case of C1-BODIPY-C12-COOH, there is asigni¢cant increase in the ratio of quenching by the5-SLPC relative to 12-SLPC at high pH without anincrease in quenching by TempoPC for both C8-BODIPY-C5-COOH and C10-BODIPY-C3-COOH(Tables 3 and 4). This would be predicted if therewas a deep subpopulation with a depth that de-creases upon COOH ionization and a shallow pop-ulation that does not decrease in depth upon ioniza-tion.

In further support of this idea the pH dependenceof the quenching of C8-BODIPY-C5-COOH fattyacid shows a change in depth at the pH at whichthe COOH group ionizes (i.e. pKa) (Fig. 3). Thepopulation of molecules with a deep BODIPY grouphas COOH pKa close to 7 which is similar to thatpreviously observed for a number of derivatives offree fatty acids [23,24]. This also suggests behaviorthe deep population of BODIPY fatty acids is notperturbed by the BODIPY group.

Fig. 4. The dependence of zcf on emission wavelength. zcf wasmeasured as a function of wavelength for: (a) C10-BODIPY-C3-COOH; and (E) C8-BODIPY-C5-COOH at pH 10. (999)emission spectrum of C8-BODIPY-C5-COOH at pH 10. Emis-sion spectra of the di¡erent BODIPY derivatives were very sim-ilar.

Fig. 3. The pH dependence of zcf apparent for the deep subpo-pulation of C8-BODIPY-C5-COOH. Note that zcf apparent isthe upper limit to the true zcf of the deep population in thiscase (see Section 2). The change in zcf relative to that at pH 4is shown on the y-axis.

BBAMEM 77445 24-9-98


3.6. E¡ect of cholesterol and emission wavelengthon zcf

We also examined the e¡ect of cholesterol on themembrane location of the BODIPY probes. Asshown by a comparison of Tables 2 and 4 to Tables1 and 3 there was little signi¢cant di¡erence betweenBODIPY depth in membranes lacking or containing33% cholesterol.

Previous studies showed that the apparent depth ofsome, but not all, £uorescent probes is dependent onthe measured emission wavelength [5,20,24]. To de-termine whether this is a feature of BODIPY probes,we investigated the apparent depth dependence oftwo BODIPY fatty acids on emission wavelength.As shown in Fig. 4 there was no signi¢cant depend-ence of apparent depth on emission wavelength.

4. Discussion

4.1. BODIPY acts like a polar membrane probe, butis not as polar as NBD or dansyl probes

This study has provided a relatively direct meas-urement of the location of the BODIPY probes inmembranes. This is important because it will allowmore exact interpretation of the behavior of thesewidely used probes, and will help in developing rulesfor predicting the relationship of chemical structureto membrane depth [5,6,20].

We ¢nd that the BODIPY group has a clear ten-dency to locate in the polar headgroup region of thebilayer. The main population of BODIPY probeslocate at a depth similar to that seen previously forsuch polar £uorophores as NBD [2,3] and dansyl [20]derivatives. This does not mean that the BODIPYgroup is as polar as NBD or dansyl, because ahigh degree of polarity is not necessary for a groupto have a tendency to locate shallowly. For example,carbazole only has three fused rings containing onenitrogen atom, yet has a signi¢cant tendency to lo-calize near the depth of the polar/hydrocarbon boun-dary [6,25].

In fact, BODIPY has a lesser tendency to locatewithin the polar headgroup region than NBD anddansyl. This is shown by the observation that, unlikeboth NBD and dansyl fatty acid derivatives, there

was a deep population of BODIPY groups whenBODIPY was attached to the end of a fatty acylchain. Although the presence of a predominatingshallow population makes it di¤cult to determinethe precise depth of the deeper population, the upperlimit for the depth of the deeper BODIPY popula-tion (6 7.5 Aî ) suggests the conformation of the acylchain is near normal when the BODIPY locatesdeeply. Furthermore, the location of this deeper pop-ulation responds to ionization of the fatty acid car-boxyl group the same way (by moving to a few Aî

shallower location) as has been found for other hy-drophobic fatty acid derivatives [23,24,26]. There-fore, we conclude BODIPY fatty acyl derivativesshould be superior analogs of natural fatty acidsthan the corresponding NBD and dansyl derivatives.

4.2. The behavior of BODIPY probes is consistentwith accessibility to iodide quenching

On the basis of the reduced accessibility of BOD-IPY derivative to quenching by KI when membranebound relative to that when dissolved in solution,Johnson et al. [14] proposed that the BODIPY groupwas deeply buried in membranes. However, this re-sult was not de¢nitive for two reasons. First, is that astrong reduction in accessibility to quenchers can oc-cur even when a £uorophore is bound to the polarheadgroup region. We found that for carbazolequenching by the aqueous quencher acrylamide thereis reduction in accessibility of a £uorophore to aquencher even upon binding to a shallow locationin membranes [6]. Furthermore, the reduction of ac-cessibility to quenching upon binding near the sur-face was much larger than the di¡erence in accessi-bility upon shifting from a shallow to a deep locationwithin the bilayer [6].

Second, the dependence of Fo/F upon [KI] data inJohnson et al showed a slope that deviated fromlinearity with a negative curvature. This is character-istic of quenching behavior when there are two pop-ulations with di¡erent accessibilities to quenching[27]. In such cases, the deep population dominatesthe slope of the Fo/F vs. [quencher] curve at highquencher concentrations. Therefore, the KI data sup-ports our conclusion that multiple populations ofBODIPY probes exist at di¡erent depths in mem-branes.

BBAMEM 77445 24-9-98


Acknowledgements

This research was supported by NIH Grant GM48596.

References

[1] A. Chattopadhyay, E. London, Parallax method for directmeasurement of membrane penetration depth utilizing £uo-rescence quenching by spin-labeled phospholipids, Biochem-istry 26 (1987) 39^45.

[2] D.E. Wolf, A.E. Ting, K.M. Bocian, R.E. Pagano, determi-nation of the transbilayer distribution of £uorescent lipidanalogues by nonradiative resonance energy transfer, Bio-chemistry 31 (1992) 2865^2873.

[3] F.S. Abrams, E. London, Extension of the parallax analysisof membrane penetration depth to the polar region of modelmembranes: use of £uorescence quenching by a spin-labelattached to the phospholipid polar headgroup, Biochemistry32 (1993) 10826^10831.

[4] F.S. Abrams, E. London, Calibration of the parallax £uo-rescence quenching method for determination of membranepenetration depth: re¢nement and comparison of quenchingby spin-labeled and brominated lipids, Biochemistry 31(1992) 5312^5322.

[5] E. Asuncion-Punzalan, E. London, Control of the depth ofmolecules within membranes by polar groups: determinationof the location of anthracene-labeled probes in model mem-branes by parallax analysis of nitroxide-labeled phospholipidinduced £uorescence quenching, Biochemistry 34 (1995)11460^11466.

[6] K. Kachel, E. Asuncion-Punzalan, E. London, Anchoring oftryptophan and tyrosine analogs at the hydrocarbon-polarboundary in model membrane vesicles : parallax analysis of£uorescence quenching induced by nitroxide-labeled phos-pholipids, Biochemistry 34 (1995) 15475^15479.

[7] J. Ren, S. Lew, Z. Wang, E. London, Transmembrane ori-entation of hydrophobic K-helices is regulated by a physio-logically relevant sequence lengths and cholesterol concen-trations, Biochemistry 36 (1997) 10213^10220.

[8] L.R. Palmer, A.R. Merrill, Mapping the membrane topologyof the closed state of the colicin E1 channel, J. Biol. Chem.269 (1994) 4187^4193.

[9] T.S. Ramalingam, K.D. Puspendu, S.K. Podder, Ricin-mem-brane interaction: membrane penetration depth by £uores-cence quenching and resonance energy transfer, Biochemis-try 33 (1994) 12247^12254.

[10] L.A. Chung, J.D. Lear, W.F. De Grado, Fluorescence stud-ies of the secondary structure and orientation of a model ionchannel peptide in phospholipid vesicles, Biochemistry 31(1992) 6608^6616.

[11] J.D. Jones, L.M. Gierasch, E¡ect of charged residue substi-tutions on the membrane-interactive properties of signal se-quences of the Escherichia coli LamB protein, Biophys. J. 67(1994) 1534^1545.

[12] R.P. Haugland, in: K.D. Larison (Ed.), Handbook of Fluo-rescent Probes and Research Chemicals, 6th edn., MolecularProbes, Eugene, OR, 1996.

[13] J. Karolin, L.B.A. Johansson, L. Strandberg, T. Ny, Fluo-rescence and absorption spectroscopic properties of BODI-PY derivatives in liquids, lipid membranes and proteins,J. Am. Chem. Soc 166 (1994) 7801^7806.

[14] I.D. Johnson, H.C. Kang, R.P. Haugland, Fluorescent mem-brane probes incorporating dipyrrometheneboron di£uoride£uorophores, Anal. Biochem. 198 (1992) 228^237.

[15] R.C.A. Keller, J.R. Silvius, B. DeKruij¡, Characterization ofthe resonance energy transfer couple coumarin-BODIPY andits possible applications in protein-lipid research, Biochem.Biophys. Res. Commun. 207 (1995) 508^514.

[16] O.C. Martin, R.E. Pagano, Internalization and sorting of a£uorescent analogue of glucosylceramide to the Golgi appara-tus of human skin ¢broblasts, J. Cell Biol. 125 (1994) 769^778.

[17] S. Frey, M. Marsh, S. Gunther, A. Pelchen-Matthews, P.Stephens, S. Ortlepp, T. Stegmann, Temperature dependenceof cell^cell fusion induced by the glycoprotein of human im-munode¢ciency virus type 1, J. Virol. 69 (1995) 1462^1472.

[18] R.J. Martin, J.R. Kusel, On the distribution of a £uorescentivermectin probe in Ascaris membranes, Parasitology 104(1992) 549^555.

[19] Y. Deng, J.R. Bennink, H.C. Kang, R.P. Haugland, J.W.Yewdell, Fluorescent conjugates of brefeldin A selectivelystain the ER and Golgi complex of living cells, J. Histochem.Cytochem. 43 (1995) 907^915.

[20] E. Asuncion-Punzalan, K. Kachel, E. London, Groups withpolar characteristics can locate at both shallow and deeplocations in membranes: the behavior of dansyl and relatedprobes, Biochemistry 37 (1998) 4603^4611.

[21] D.M. Engelman, J.E. Rothman, The planar organization oflecithin-cholesterol bilayers, J. Biol. Chem. 247 (1972) 3694^3697.

[22] J.H. Ipsen, O.G. Mouritsen, M. Bloom, Relationships be-tween lipid membrane area, hydrophobic thickness, andacyl-chain orientational order: the e¡ects of cholesterol, Bi-ophys. J. 57 (1990) 405^412.

[23] R.D. Kaiser, E. London, Location of diphenylhexatriene(DPH) and its derivatives within membranes: comparisonof di¡erent £uorescence quenching analyses of membranedepth, Biochemistry 37 (1998) 8180^8190.

[24] F.S. Abrams, A. Chattopadhyay, E. London, Determinationof the location of £uorescence probes attached to fatty acidsusing parallax analysis of £uorescence quenching: e¡ect ofcarboxyl ionization state and environment on depth, Bio-chemistry 31 (1992) 5322^5327.

[25] W.C. Wimley, S.H. White, Membrane partitioning: distin-guishing bilayer e¡ects from the hydrophobic e¡ect,Biochemistry 32 (1993) 6307^6312.

[26] M.D. Barnett, P. Laggner, The pH-dependence of ESR spec-tra from nitroxide probes in lecithin dispersions, Biochim.Biophys. Acta 363 (1974) 127^133.

[27] M.R. Eftink, C.A. Ghiron, Fluorescence quenching studieswith proteins, Anal. Biochem. 114 (1981) 199^227.

BBAMEM 77445 24-9-98


Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence...

Documents

Transcript of Determination of the depth of BODIPY probes in model membranes by parallax analysis of fluorescence...