long-lifetime lipid probe containing a luminescent metal-ligand complex

5
Long-Lifetime Lipid Probe Containing a Luminescent Metal – Ligand Complex LI LI, HENRYK SZMACINSKI, JOSEPH R. LAKOWICZ Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201 Received 17 September 1996; revised 9 September 1996; accepted 15 October 1996 ABSTRACT: We describe the chemical synthesis and spectral properties of a long-life- time luminescent probe for membranes. A ruthenium metal – ligand complex was cova- lently coupled to the amino group of phosphatidyl ethanolamine. When incorporated into model membranes, this probe displays decay times near 500 ns. Importantly, the probe displays polarized emission and can be used to study membrane motions on the microsecond timescale. q 1997 John Wiley & Sons, Inc. Biospect 3: 155–159, 1997 Keywords: fluorescence; metal–ligand probes; long lifetime probes; polarization; anisotropy; lipids; membranes INTRODUCTION ing phosphorescence. 5–7 However, the use of phos- phorescence requires the complete exclusion of ox- ygen, and the phosphorescence time-zero anisot- Cell membranes are predominantly composed of ropies are often low. phospholipids, which are spectroscopically silent In this communication we describe an alterna- in the ultraviolet and visible regions of the spec- tive method to obtain luminescent probes with trum. Consequently, extrinsic fluorophores em- long decay times. We synthesized a phospholipid bedded in membranes or attached to the phospho- analogue which contains a covalently attached lipids are typically used to study the structure metal–ligand complex (MLC) (Scheme 1). Such and dynamics of membranes. 1,2 The physical prop- complexes are known to display decay times near erties of membranes have been studied using en- 400 ns and polarized emission. 8,9 Hence, we expect ergy transfer, solvent-sensitive fluorophores, and the conjugate of Ru(bpy) 2 (mcbpy) (Ru-PE) anisotropy measurements. However, the vast ma- where bpy is 2,2 *-bipyridine, mcbpy is 4-carboxy- jority of membrane probes display decay times of 4 *-methyl-2,2 *-bipyridine, and PE is dipalmitoyl- 1–10 ns, which limits the information content of L-a-phosphatidyl ethanolamine, to be a long-life- the measurements to phenomena which can affect time lipid probe providing the opportunity to mea- the excited state on this timescale. Consequently, sure microsecond motions based on its polarized with the exception of studies using pyrene 3 and emission. fluorescence recovery after photobleaching, 4 most fluorescence experiments are not able to reveal the rates of lipid diffusion in bilayers or phenom- ena on the microsecond timescale. The limitations MATERIALS AND METHODS of short decay times have been circumvented us- Synthesis of Ru-PE Correspondence to: J. R. Lakowicz. The synthesis of [Ru(bpy) 2 (mcbpy)] (PF 6 ) 2 has Contract grant sponsor: National Institutes of Health; con- been described previously. 8 A total of 360 mg of tract grant numbers: GM-35154 and RR-08119. q 1997 John Wiley & Sons, Inc. CCC 1075-4261/97/020155-05 Ru(bpy) 2 ( mcbpy ) ( PF 6 ) 2 and 51 mg of N-hydroxy- 155 96-041 / 8W06$$6041 02-26-97 09:28:10 biosa W: Bio Spec

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Page 1: Long-lifetime lipid probe containing a luminescent metal-ligand complex

Long-Lifetime Lipid Probe Containing a LuminescentMetal–Ligand Complex

LI LI, HENRYK SZMACINSKI, JOSEPH R. LAKOWICZ

Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of MarylandSchool of Medicine, 108 North Greene Street, Baltimore, Maryland 21201

Received 17 September 1996; revised 9 September 1996; accepted 15 October 1996

ABSTRACT: We describe the chemical synthesis and spectral properties of a long-life-time luminescent probe for membranes. A ruthenium metal–ligand complex was cova-lently coupled to the amino group of phosphatidyl ethanolamine. When incorporatedinto model membranes, this probe displays decay times near 500 ns. Importantly, theprobe displays polarized emission and can be used to study membrane motions on themicrosecond timescale. q 1997 John Wiley & Sons, Inc. Biospect 3: 155–159, 1997

Keywords: fluorescence; metal–ligand probes; long lifetime probes; polarization;anisotropy; lipids; membranes

INTRODUCTION ing phosphorescence.5–7 However, the use of phos-phorescence requires the complete exclusion of ox-ygen, and the phosphorescence time-zero anisot-Cell membranes are predominantly composed ofropies are often low.phospholipids, which are spectroscopically silent

In this communication we describe an alterna-in the ultraviolet and visible regions of the spec-tive method to obtain luminescent probes withtrum. Consequently, extrinsic fluorophores em-long decay times. We synthesized a phospholipidbedded in membranes or attached to the phospho-analogue which contains a covalently attachedlipids are typically used to study the structuremetal–ligand complex (MLC) (Scheme 1). Suchand dynamics of membranes.1,2 The physical prop-complexes are known to display decay times nearerties of membranes have been studied using en-400 ns and polarized emission.8,9 Hence, we expectergy transfer, solvent-sensitive fluorophores, andthe conjugate of Ru(bpy)2(mcbpy) (Ru-PE)anisotropy measurements. However, the vast ma-where bpy is 2,2 *-bipyridine, mcbpy is 4-carboxy-jority of membrane probes display decay times of4 *-methyl-2,2 *-bipyridine, and PE is dipalmitoyl-1–10 ns, which limits the information content ofL-a-phosphatidyl ethanolamine, to be a long-life-the measurements to phenomena which can affecttime lipid probe providing the opportunity to mea-the excited state on this timescale. Consequently,sure microsecond motions based on its polarizedwith the exception of studies using pyrene3 andemission.fluorescence recovery after photobleaching,4 most

fluorescence experiments are not able to revealthe rates of lipid diffusion in bilayers or phenom-ena on the microsecond timescale. The limitations MATERIALS AND METHODSof short decay times have been circumvented us-

Synthesis of Ru-PECorrespondence to: J. R. Lakowicz. The synthesis of [Ru(bpy)2(mcbpy)] (PF6)2 hasContract grant sponsor: National Institutes of Health; con-

been described previously.8 A total of 360 mg oftract grant numbers: GM-35154 and RR-08119.q 1997 John Wiley & Sons, Inc. CCC 1075-4261/97/020155-05 Ru(bpy)2(mcbpy) (PF6)2 and 51 mg of N-hydroxy-

155

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156 LI, SZMACINSKI, AND LAKOWICZ

was cavity dumped at 1.5099 MHz. The emissionabove 610 nm was isolated using a Corning 2-61filter. The intensity decays were fit to a singleexponential decay law

I (t ) Å I0 e0t /t (1)

where I0 is the time-zero amplitude and t the de-cay time. The anisotropy decays were fit to

r (t ) Å ∑2

iÅ1

ri e0t /ui / r` (2)

where ri are the amplitudes of the anisotropy de-caying with the correlation times ui , and r` is theapparent anisotropy at long times. The parameterScheme 1. Chemical structure of Ru(bpy)2(mcbpy)-values were recovered by nonlinear least squaresPE (Ru-PE).using software from IBH, Inc.

Preparation of Lipid Vesiclessuccinimide (NHS) were dissolved in 1.2 mL of

Lipid vesicles were prepared by dissolving dipal-acetonitrile at room temperature. Then, 120 mg ofmitoyl-L-a-phosphatidyl glycerol (DPPG) and Ru-N,N*-dicyclohexylcarbodiimide (DCC) was added.PE at a 50 : 1 molar ratio in CHCl3/MeOH (95/The mixture was sealed and stirred for several5, v/v), and the solvent was removed by a streamhours. The precipitate was removed by filtrationof argon while the solution was kept at 557C. Vesi-through a syringe filter. The filtrate was added tocles were prepared by sonication in 10 mM Trisstirred solution of 2-propanol, and the mixtureand 50 mM KCl, pH 7.5, at a final lipid concentra-was kept at 047C for 1 h. The precipitate, Ru(b-tion of 2 mg/mL. The DPPG vesicles in the ab-py)2(mcbpy)-NHS, was removed by filtration andsence of Ru-PE did not display significant signalswashed with dry ether (3 1 5.5 mL).under the present experimental conditions. Fluo-Conjugation of the NHS ester to PE was accom-rescence measurements were performed in equi-plished by adding 120 mg of Ru(bpy)2(mcbpy)-librium with the air.NHS in 4.5 mL of dry dimethyl formamide (DMF)

to 80 mg of PE. The PE was dissolved in 10 mLRESULTSof CHCl3 with 6 mL of triethylamine. The mixture

was stirred for 20 h in the dark under an argon Absorption and emission spectra of DPPG vesiclesatmosphere. The solvents were removed under labeled with Ru-PE are shown in Figure 1. Thevacuum and the product was redissolved in 2.5mL of CHCl3/MeOH (2 : 1, v/v). Pure Ru-PE wasobtained by thin-layer chromatography on K6Fsilica gel plates using CHCl3/MeOH/H2O (65 : 25 :4, v/v/v) as the developing solvent, with (PF6) asthe counter ion. The Rf value of the product isnear 0.6, relative to that of PE (0.78). We notethat the product Ru-PE has a net charge of /1.For simplicity, this charge is assumed to be pres-ent without explicit mention.

Instrumentation

Emission spectra were recorded on a SLM AB2spectrofluorometer (Spectronic, Rochester, NY).The time-domain intensity and anisotropy decays Figure 1. Absorption and emission of Ru(bpy)2were obtained by time-correlated single-photon (mcbpy)-PE in DPPG vesicles. The anisotropy spec-counting.10 The excitation source was a frequency- trum (dashed line) is for Ru(bpy)2(mcbpy) in glycerol–

water (6/4, v/v) at 0557C.doubled Pyridine 1 dye laser at 360 nm, which

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LONG-LIFETIME LIPID PROBE 157

instrument for time-correlation single-photoncounting, which is designed for measurement ofshorter nanosecond decay times. While the lim-ited time range may have prevented us from ob-serving a possible multiexponential intensity de-cay, it is clear from the data that the overall decaytimes of Ru-PE are long.

For use as an anisotropy probe, the metal–li-gand complex must display polarized emission.The excitation anisotropy spectra of the parentcompound Ru(bpy) (mcbpy)/1 is shown in Figure1. In the absence of rotational diffusion in glyc-erol–water (6/4; v/v) at 0557C, this complex dis-plays a maximal anisotropy of 0.17. We examinedthe anisotropy spectrum of this complex, insteadof Ru-PE, because of poor solubility of Ru-PE inglycerol–water. In previous studies,8,9 we foundthat the excitation anisotropy spectra were simi-lar for the MLC probes as the free carboxylic acidsor when covalently linked to the amino groups of

Figure 2. Intensity decays of Ru-PE in DPPG vesi- proteins. Hence, we expect the anisotropy spec-cles. trum of the parent MLC to reflect that of the lipid

probe. While the maximal anisotropy of Ru-PE(0.17) is smaller than observed for other metal–

absorption maximum near 450 nm is well sepa- ligand complexes, this value is adequate for an-rated from the emission maximum near 630 nm. isotropy measurements.Such a large Stokes shift is an advantage for a

Time-resolved anisotropy decays of Ru-PE inlipid probe, because membrane suspensions typi-DPPG vesicles are shown in Figure 3. As for thecally scatter light, which is more easily removedintensity decays, we show the fits to the time-when the excitation wavelength is displaced fromresolved anisotropy data (Fig. 3). These datathe emission wavelength. The emission spectrumwere obtained with an excitation wavelength ofis insensitive to temperature from 27C to 507C,360 nm, which does not correspond to the maxi-except for a decreasing intensity with increasingmum value of the anisotropy, but rather to antemperature. At 257C the quantum yield of Ru-anisotropy value near 0.07 (Fig. 1). This excita-PE in DPPG vesicles is similar to that of Ru(bpy)3tion wavelength was the longest wavelengthin aqueous solution. More specifically, the quan-available from our dye lasers. Despite the lowtum yield is near 0.044, as determined relative totime-zero anisotropy (0.07 at 360 nm excitation),that of Ru(bpy)2/

3 in aqueous solution with anwe were able to recover the anisotropy decaysassumed quantum yield of 0.042.11

(Fig. 3). Visual inspection of the data reveals aIntensity decays of Ru-PE–labeled vesicles arerapid correlation time õ 11 ns and the presenceshown in Figure 2. Because of the low emissionof a nonzero anisotropy value (r` ) at long times.rate of the Ru complex, the time-resolved inten-Least-squares analyses of these data reveal thesity decays contained relatively few counts andpresence of two or more correlation times (5–11thus high Poisson noise. Hence, Figure 2 showsand 47–124 ns), as well as a nonzero value ofthe fits to the data, which are adequate to showr` . The total anisotropy recovered for each decaythe long decay time. The intensity decays ap-(Table I) was similar to that expected for 360-peared to be a single exponential, with decaynm excitation. While nonzero r` values have oftentimes ranging from 606 ns at 47C to 369 ns atbeen observed with nanosecond fluorophores,12,13467C (Table I) . Therefore, the Ru-PE probe dis-the values in Table I should be regarded as pre-plays the desired property of a long decay time.liminary data. We note that the time range of ourExamination of Figure 2 reveals that the pres-measurements was limited to 250 ns, and that theent measurements were performed over a limitedr` values may be apparent values resulting fromtimescale, less than a single decay time. The lim-

ited time range reflects the capabilities of our the limited time range of the measurements.

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158 LI, SZMACINSKI, AND LAKOWICZ

Table I. Lifetimes and Anisotropy Decay Analysis of Ru(bpy)2(mcbpy)-PE-labeled DPPG Vesicles

T (7C) t (ns) ui (ns) ri r` rTOTa x2

R

4 606 61.3 0.030 0.027 0.057 1.12311.0 0.017

123.7 0.025 0.023 0.065 1.10320 480 36.4 0.040 0.013 0.053 1.069

4.5 0.02046.9 0.030 0.013 0.063 1.055

46 369 9.0 0.061 0 0.061 1.042

a rTOT Å r1 / r2 / r` . The measured anisotropy in the absence of rotational diffusion for 360 nm excitation is 0.07 (Fig. 1).

The authors acknowledge the National Institutes ofDISCUSSIONHealth (Grants GM-35154 and RR-08119) for supportfor this research.In previous reports we described the usefulness of

MLCs as covalent probes for microsecond proteindynamics.8,9 It is important to recognize thatMLCs can display a variety of spectral properties. REFERENCESFor instance, by selection of the metal or ligand,one can obtain probes with absorption from 600 1. J. Slavik, Ed. Fluorescent Probes in Cellular andto 700 nm.14–16 It is also possible to increase the Molecular Biology, CRC Press, Boca Raton, Flor-quantum yields to near 0.5 and the decay times ida, 1994.to over 10 ms by choice of the ligands.17–19 These 2. T. G. Dewey, ‘‘Fluorescence energy transfer in

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