Diphenylhexatrienylpropanolylhydrazyl stachyose: a new oligosaccharide derivative of diphenylhexatriene. Synthesis and fluorescence properties in artificial membranes

Chemistry and Physics of Lipid.s, 49 (1988) 185-195 Elsevier Scientific Publishers Ireland Ltd.

185

Diphenylhexatrienylpropanoylhydrazyl stachyose: a new oligosaccharide derivative of diphenylhexatriene. Synthesis and fluorescence properties in artificial membranes E.N. Ivessa, E. Kalb, F. Paltauf and A. Hermetter Department of Biochemisa'y, Graz University of Technology, A-8010 Graz (Austria) (Received May 2nd, 1988; revised and accepted August 22nd, 1988) Condensation of diphenylhexatrienylpropanoyl hydrazide with stachyose oxidized to an aldehyde at C6 of the terminal galactose, followed by reduction with sodium borohydride yields diphenylhexatrienylpropanoylhydrazyl stachyose (glyco-DPH). This new fluorescence probe inserts almost instantaneously into artificial phospholipid vesicles and biological membrane~. Due to its large hydrophific carbohydrate portion, it serves as an impermeant, uncharged probe with a defined orientation within the membrane bilayer. Its usefulness to monitor lipid mobility was proven by measuring fluorescence anisotropies of dipalmitoylglycerophospbocholine at temperatures around the gel to liquid phase transition, and by measuring the rigidifying effect of cholesterol on egg yolk phosphatidylcholine membranes. Fluorescence lifetimes of glyco-DPH are best fitted by bimodal Lorentzian distributions. A predominant lifetime component centered at 4.3 ns in ethanol and at 6.1 ns in vesicles of 1-palmitoyl-2-oleoyl-snglycero-3-phosphocboline (POPC) is obtained, with a narrower distribution within POPC, showing that glyco-DPH is distributed more homogeneously in the phospbolipid membrane.

Keywords: fluorescence anisotropy; fluorescence lifetime distributions; membrane fluidity.

Introduction Diphenylhexatriene ( D P H ) [1-3] and its amphiphilic analogue trimethylammoniumdiphenylhexatriene ( T M A - D P H ) [4,5] are widely used probes in m e m b r a n e research. T h e i r fluorescence anisotropies respond to local changes of motion and orientation of their lipid surroundings in an artificial or biological m e m brane, though to a different extent. T M A - D P H is oriented parallel to the m e m b r a n e normal and "fixed" to the hydrophobic-hydrophilic m e m brane interface via its charged head group. In contrast, the totally apolar D P H resides in the apolar m e m b r a n e interior and m a y exhibit, in addition, orientations different from the m e m brane normal. T h e r e f o r e , it possesses m o r e d e g r e e s of motional freedom c o m p a r e d with

Correspondence to: A. Hermetter.

T M A - D P H [6] and, therefore, responds m o r e significantly to changes in bilayer lipid mobility with regard to its fluorescence anisotropy. T h e r e fore, D P H m a y be preferentially used for "fluidity" studies on artificial m e m b r a n e s or defined (intra-)cellular m e m b r a n e fractions. Nevertheless, one has to take into account that the label orientation of D P H in a m e m b r a n e is illdefined, and actually it is uncertain what anisotropy changes do mean, if m e m b r a n e fluidity changes of e.g. complex biological m e m b r a n e s are to be determined. A new p r o b l e m arises if this label is considered for m e m b r a n e studies on whole living cells. F r o m fluoresence microscopy studies it is known that e v e n under various labeling conditions, D P H distributes a m o n g the entire cell [7]. In the worst case, D P H may not only be associated with m e m b r a n e o u s structures but also with proteins or lipid droplets (containing triglyceride) within the cells. On the other hand, it is possible to label selectively cell surfaces with T M A - D P H

0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

186

under certain circumstances and to study plasma membrane fluidities of living cells in a defined way [5,7]. TMA-DPH is a cationic probe and, therefore, it cannot be excluded that it may associate preferentially with particular lipid or protein domains in the biomembrane that are negatively charged. Furthermore, TMA-DPH resides in the surface membrane of cultured cells only for a limited time eventually entering the cell [7]. Therefore, it was desirable to have an amphiphilic probe which is photophysically suited for membrane lipid mobility studies, with defined orientation in the bilayer or a cell membrane, electroneutral head group and favourable properties in maintaining its localisation once inserted into the plasma membrane of a cell. Cogan and Schachter [8] synthesized pyrenebutanoylhydrazyl stachyose, a sugar conjugate of pyrenebutanoic acid. Due to its large polar head group it was suggested as a non-permeant membrane probe for defined membrane studies on biological systems. However, fluorescence anisotropies are very small due to the very long pyrene fluorescence lifetime (~100 ns) and anisotropy changes are hard to detect in a reliable manner. Though this system basically looked very promising, it was necessary to apply it to a fluorophore with more suitable photophysics and sufficiently high chemical stability. Therefore, we decided to synthesize glyco-DPH, a new oligosaccharide conjugate of DPH-propanoic acid which fulfills the above mentioned criteria. In this paper we describe its chemical synthesis as well as its fluorescence characteristics (spectra, anisotropies and lifetimes) in artificial membranes of different fluidity state and cholesterol content. In two separate papers, its application to fluidity studies on cultured human skin fibroblasts and intracellular membranes from yeast will be reported.

Materials a~l methods Materials DPH was purchased from Serva (Heidelberg, F.R.G.), TMA-DPH and 3-DPH-propanoic acid were from Molecular Probes (Eugene, Oregon).

Phosphatidylcholine from egg yolk was obtained from Lipid Products (Nutfield, U.K.). Cholesterol was from Merck (Darmstadt, F.R.G.) and was further purified by recrystallization from methanol/water. Galactose oxidase (EC 1.1.3.9) from Dactylium dendroides, staehyose and Triton X-100 were from Sigma (Deisenhofen, F.R.G.). Silica gel 60, PF254 as well as all other chemicals used were p.a. grade from Merck. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) were from Lucas Meyer (Hamburg, F.R.G.).

Preparation of glyco- DPH DPH-pmpanoyl methylester DPH-propanoic acid (10mg; 33/~mol) was esterified with 1.5ml BF3.CHaOH under an atmosphere of argon (100/~1 benzene was added to increase solubility) for 20 rain at 95"C in an oil bath [9]. The product was extracted with benzene, washed with aqueous bicarbonate, and dried over sodium sulphate. The reaction was quantitative.

DPH-propanoyl hydrazide The DPH-propanoic methylester was mixed with 300/xl ethanol, 150/~l 80% (3.1 retool) hydrazine hydrate and 25/zl benzene. The reaction was carried out under an atmosphere of argon, either for 3 h at 105 °C in an oil bath or overnight at 60"C with stirring [10]. After addition of 750 ttl H20, the product was extracted with CHCI3/CH3OH (2:1, v/v), extensively washed with CH3OH/H20/CHCIa (48:47:3, by vol.) and 1 2 0 to remove excess hydrazine, and then dried. The reaction yields are 70--80%.

Stachyose derivative of DPH-pmpanoyi hydrazide (according to the procedure of Cogan and Schachter [8]) During the first step, the hydroxy group at C6 of the terminal galactose residue was enzymatically oxidized to an aldehyde. Stachyose (50rag; 67/~mol) was dissolved in 800 ttl 0.1 M potassium phosphate buffer (pH 6.0); 20 units of galactose oxidase (EC 1.1.3.9)from Dactylium dendmides dissolved in 200 ~1 buffer were added. The reac-

187

tion was carded out at 25°C for 2 h with slight stirring. During the next step, the selectively oxidized sugar was coupled with the DPH-propanoyl hydrazide prepared as described above. The stachyose solution was adjusted to pH 5.6 with 0.1 M HCI and the DPH-propanoyl hydrazide suspended in 800/~l tetrahydrofuran was added. The reaction mixture was stirred at 37°C for 3 h followed by stirring at 25°C overnight. The last step comprises the reduction of the Schiff's base. The reaction mixture was adjusted to pH 8.3 with 1 M N a O H and cooled on ice; 10 mg NaBH4 dissolves in 100/~l ice-cold 0.1 M potassium phosphate buffer (pH 8.0) was added. The reaction was kept at 25 °C for 30 rain and then at 4°C for 4 h. The pH was lowered to 5 with glacial acetic acid and the mixture concentrated on a rotavapor at 35 °C to a small volume. A precipitate formed which was extracted with CH3OI-I/H20 (1:1, v/v), and after short centrifugation in a labofuge, the extracted material was purified by thin-layer chromatography on 0.5 mm thick silica gel plates. The plates were developed by two separate runs, first with CHCI3/CH3OH (9:1, v/v) to remove unreacted fluorescent material from the product, which remains at the origin, and then in the same direction with n-butanol/acetic acid/H20 (4:1:2, by vol.), where the product moves with an Rf value of approximately 0.35. The fluorescent band was scraped off the plate, and the product eluted from the silica gel with C H 3 O H / H 2 0 (1:1, v/v). The fluorophor concentration of the product was determined from the absorbance in tetrahydroO HN

HN~CH2

r----- Cllz

| I o ,,o /

I

xo ! I

OX

I

CH2 OH

/

OH

~

cx ox

OH

I Cx2ox

OH

Fig. L Structure of diphenylhexatrienylpropanoylhydrazyl

stachyose (glyco-DPH).

furan at 360 nm; D P H propanoic acid was used as standard. The sugar content of the product was analyzed by the phenol-sulfuric acid method [11]. The DPH/stachyose ratio was 0.7. The overall product yield based on the D P H content was approximately 10% relative to D P H propanoic acid. Figure 1 shows the structure of glyco-DPH. a

Preparation o[ lipid vesicles Appropriate amounts of egg yolk phosphatidylcholine (egg PC) or POPC (in CHCIa/CH3OH 2:1, v/v) and cholesterol (in CHCI3) were mixed and dried under a stream of nitrogen. The lipids were suspended in 10 mMTris-HCl buffer (pH 7.4) to give a final lipid concentration of 0.2 mM (egg PC) or 2.5 mM (POPC), respectively, and sonicated with a Brown Labsonic 2000 sonicator at 80 W for 8 min under a stream of nitrogen. Suspensions containing unsaturated lipids were sonicated under cooling in an ice-bath. Aqueous DPPC suspensions (0.5 mM) were sonicated at 45°C. Vesicles were centrifuged for 10min at 1000 g in a labofuge. Vesicle suspensions containing unsaturated or fully saturated phospholipids were stored over night at 4°C and 42°C, respectively, before use.

Fluorescence measurements Spectra and steady state fluorescence anisotropies of DPH-labels were determined on a Shimadzu RF-540 spectro-fluorometer (Kyoto, Japan) or a spectrofluorometer G R E G 200 from I.S.S. (La Spezia, Italy). Constant temperatures of the samples in quartz cuvettes were maintained by an external water bath. The samples were brought to the highest temperature applied within a series of temperature-dependent measurements. Then, 0.6--1.5/~l of 1 mM stock solutions of DPH (in tetrahydrofuran) or T M A - D P H (in ethanol) or 2.4 6.0/~l 0.25 mM stock solution of glycoD P H (in methanol/water, 1:1, v/v) were added to give final label concentrations of 0.2~0.5/zM. The lipid to label ratio was always 1000:1. Incubation times did not exceed 30rain. The fluorescence background due to residual fluoro-

188

phor in the aqueous incubation medium could be neglected as the fluorescence intensity of DPH derivatives in water is very low. For recording the emission spectra, the excitation wavelength was 360nm; monochromator slits were 10 nm and 5 nm for the excitation and the emission path, respectively. For recording the excitation spectra, the emission wavelength was 430nm; monochromator slits were 5 n m and 10 nm, respectively. Fluorescence anisotropies • [12] were determined according to r = ( I v v - G . I v n ) l ( I v v + 2 " G" Ivn) with G = I n v / l n n , I v v and I v n being the fluorescence intensities parallel and normal relative to the vertically oriented excitation polarizers. G is the gfactor. IHv and Inn are the fluorescence intensities determined with the emission polarizer oriented vertically and horizontally when the excitation polarizer was set in the horizontal position. Excitation wavelength was 360nm and emission wavelength was recorded at 430nm. Excitation and emission slits were 5 and 20 nm, respectively. The fluorescence background of a blank sample (unlabeled vesicles) never exceeded 5% of the fluorescence signal of samples containing DPH, TMA-DPH, or glyco-DPH. Uptake of glyco-DPH by egg yolk phosphatidylcholine vesicles or cells was determined from the dequenching of the label fluorescence at 430 nm. In principle, the same procedure has been used with fibroblasts and yeast spheroplasts as will be described in more detail elsewhere. Fluorescence lifetime (~') measurements of glyco-DPH in ethanol or POPC vesicles were performed with a variable frequency fluorometer G R E G 200 [13] from I.S.S. (La Spezia, Italy) ranging from I to 200 MHz. 1,4-Di-[2-(5-phenyloxazolyl)]-benzene (POPOP) served as a lifetime reference [14]. A helium-cadmium laser was used as an excitation light source (EX-wavelength = 325 rim) and a Schott KV 370 filter was used in the emission path. All measurements were carried out at 20°C. Phase and modulation data obtained at 10 different modulation frequencies were analyzed for a sum of discrete lifetime exponentials (one-, two-, or three-component fits) [ 15] as well as in terms of unimodal and bimodal Lorentzian lifetime distributions [ 16,17] characterized by

a lifetime 'Feenterand the respective full width at half maximum (FWHM). A program from I.S.S. (La Spezia, Italy) was used for the discrete and distributional lifetime fits. Theory (see Refs. 12, 15--17) Phase shifts (P) and modulation M (AC/DC ratio) of the fluorescence relative to the excitation are defined by P =

tan-' [S(to)/G(~)]

M2 = $2(o~)+ G2(to)

to is the angular frequency of light modulation. The functions S(to) and G(to) are for the discrete lifetime approach

S(o) = ~ ~ .,o/(1 + .,2o2) o(~,)

= Y.

~(1

+

~'~/.,b

The fractions ~--ain't (z~/~ = 1) represent the contribution of the lifetime component ~-, to the total fluorescence intensity of a given fluorophor; ai is the pre-exponential factor for the decay time -~ in the time-domain according to I ( t ) = Y a~ exp ( - t/r,) ¢Z a, = 1). For the continuous model, $(¢u) and G(tu) are to be written as S(oJ) =

[f(r)~tl(1 + to2~)] d~

fo f(T) d,= 1 where f(r) is, in principle, an arbitrary function of r. We use Lorentzian lifetime distributions according to f(-r) = A / { I + [(~- C)I( W/2)2]}

The lifetime center position C and the width of the

189

distribution W are the important parameters for the fitting procedure; the constant A is obtained from the normalization condition. From a set of phase angles P and modulations M measured at different modulation frequencies, a sum of discrete lifetimes or continuous lifetime distributions are determined by minimizing X2 according to

100"

EM

I',,/i ',X. / I'/ \ I

-."

-"~50'

X~ = ~ {[(,aM J,<)/,~]2 -

+ [(M,,, - Mc)/~,,,]2}/(2n - f - 1) The suffixes c and m indicate calculated and measured values, respectively; n is the number of modulation frequencies; [ is the number of parameters; o-p and o-,, are frequency-independent standard deviations of measured phase angles and modulations, respectively. Remits

Synthesis of glyco-DPH The synthesis was carried out using a sequence of reaction steps already described by Cogan and Schachter [8] for the preparation of pyrenebutanoylhydrazyl stachyose from pyrenebutanoic acid and stachyose. DPH-propanoic acid was converted to the corresponding methylester by BF3-CH3OH. DPH-propanoyl hydrazide was obtained after hydrazinolysis of the methylester with hydrazine'H20. The hydrazide was condensed with the sugar aldehyde obtained from stachyose after treatment with galactose oxidase. The resulting Schiff base was reduced with NaBI-h to give the final product DPH-propanoyihydrazyl stachyose (glyco-DPH, Fig. 1). Giyco-DPH shows low solubility in organic apolar and polar aprotic solvents. Its solubility significantly increases upon addition of small amounts of a protic solvent (e.g. methanol). It is easily dispersed in water. Fluorescence spectra Figure 2 shows excitation (Am~ = 362 nm) and emission spectra (Amax= 430 nm) of glyco-DPH in

2

"

X,

/1 v

aoo

•

It

ioo

w

~ [nml

goo

Fig. 2. Excitation (EX) and emission (IBM) spectra of 1/.tM gly¢o-DPH in tetrahy~-ofm-~ containing 0.4 vol% methanol ( - - - - - ) and emission spectrum (EM) of glyco-DPH in egg yolk ph~phatidylcholine vesicles ( , ) at 20°C.

tetrahydrofuran containing 0.4% CH3OH. The emission maximum of the label incorporated in unilamellar vesicles of egg yolk phosphatidylcholine (Fig. 2) is very similar to the emission maximum of DPH and TMA-DPH in lipid bilayers [4]. The fluorescence of glyco-DPH in water is very low even at a concentration of 1/zM, presumably due to aggregation of the amphiphilic label. After addition of Triton X-100, the fluorescence intensity increases fiftyfold.

Uptake of glyco-DPH by vesicles and cells Incorporation of glyco-DPH in the phospholipid vesicles is complete after a few minutes. However, the incorporation rate is smaller compared with label uptake by cell surface membranes, e.g. from intact human skin fibroblasts or yeast spheroplasts. Fibroblasts incorporate glycoDPH almost instantaneously (Fig. 3). Uptake of glyco-DPH into yeast spheroplasts is rather complex. This effect will be subject lo more detailed investigations.

190 100

f\

i

1

o,3o 1 I

]

f.J

.... %'.

~o

r (DPN)

L"

r

i

(Olgco-OPII) i

2

C

_c t) U

li

0

.°5o.

\.

0,I0 ~

0 LL

0,N

t

,

,

,

,

,

,

,

30 32 34 36 30 40 42 44 46 40 50 52

[eel

temporatlwe

Fig. 4. Temperature dependence of Ihmrescence anisotropies of DPH, TMA-DPH, and glyco-DPH determined in vesicles of dipalmitoylphosphatidylcholine.

0

0

. . . . 10" t [mini

'

'

Fig. 3. Uptake of glyco-DPH by egg yolk phosplmtidylcholine vesicles (1) at 40°C, yeast spheroplasts (2) at 30°C and human skin fibroblasts (3) at 37°C.

to self-aggregation of the label at low concentrations in the bilayer can be excluded from continuous lifetime distribution studies at least in the fluid membrane state. However, it cannot be excluded for the gel state of DPPC as there is a

Steady state fluorescence anisowopies The fluorescence anisotropy of glyco-DPH was determined in unilamellar vesicles of DPPC exhibiting a gel to liquid phase transition temperature at around 40°C (Fig. 4). Glyco-DPH anisotropy detects this phase transition though it responds to a lesser extent compared with TMAD P H [4] and D P H [2,4], the latter showing the most dramatic change in anisotropy. The difference between D P H and T M A - D P H in the anisotropy change at Tc of a bilayer can be interpreted in terms of the different numbers of degrees of freedom for label motion [6]. It is lower for T M A - D P H compared with D P H as the former label is strictly oriented around the bilayer normal, whereas the totally apolar D P H may have, in addition, other orientations. In this context, glycoD P H may even be more restricted with regard to motional freedom as compared to TMA-DPH, as the label is fixed to the very large polar sugar head group. Restriction of motional changes due

0,20

,°'-I 0,22 ~

,

•

20

25

0,,,.

15

30

35 temperature

40

45

IOCl

Fig. 5. Temperature dependence of fluorescence anisotropies of glyco-DPH in vesicles of egg yolk phosphatidyicholine/cholesterol mixtures, rq, r (100/0); 0, r (90/10); IR, r

(70/30); o, r (50/50).

191 slight decrease in fluorescence intensity, probably due to self-quenching on going through Tc to lower temperatures. Increasing amounts of cholesterol in a liquid crystalline phospholipid membrane lead to a significant increase of the glyco-DPH anisotropies (Fig. 5). The influence of temperature on the anisotropy of the label in vesicles of different phospholipid to sterol ratios is almost the same except for the sterol-free membranes showing a more pronounced temperature dependence. Drastic influences of cholesterol were already reported on the fluorescence anisotropies of DPH and TMA-DPH in phospholipid bilayers [18,19]. Fluorescence lifetimes

Fluorescence lifetimes of glyco-DPH were determined in ethanol and POPC vesicles using the harmonic method. Typical sets of phase angles and modulations at different modulation frequencies between l and 200 MHz were measured and fitted to a sum of discrete exponential decays or continuous lifetime distributions (Tables I and n). In ethanol as well as in POPC vesicles, discrete fits became better, in general, on going from one to three component fits. However, only the application of a second component causes a significant decrease in X2, whereas the addition of a third component (with an insignificant fraction and a meaningless value) leads only to a smaller change in X2. The picture which emerges from a two component fit is very similar to data for DPH and TMA-DPH [6]. The major fraction of fluorescence intensity contributes to a long lifetime component (around 6.1 ns in POPC membranes) and a short component of 1.7 ns. The longer component (6.1 ns) observed in lipid bilayers is somewhat larger than the TMA-DPH lifetime (~"= 3.9 ns) but significantly smaller than the DPH lifetime (1-= 10.4 ns) in the same system at 20°C. This surprising decrease in the decay time of glyco-DPH may be attributed to the localization of the fluorphor closer to the membrane interface as well as to the presence of the polar sugar substituent in the label molecule. Differences in fluorescence lifetimes of the res-

pective DPH derivatives in ethanol suggest that the internal electronic structure effects might contribute to the lifetime differences observed in bilayer membranes to some extent. The lifetime of glyco-DPH in ethanol (4.3 ns, see Table I) is lower as compared to DPH (5.6 ns, [20]), but higher compared with TMA-DPH

(0.21 ns, [4]). Lifetimes of glyco-DPH in ethanol are lower (4.3 ns) than in POPC bilayers (6.1 ns). In ethanol, the fluorophor might be much more exposed toward the quenching effect of the solvent hydroxyls as compared to the situation in POPC bilayers where the hydrophobic fluorophor portion is shielded from solvent (H20) by the apolar acyl rest. The measured phase angles and modulations of glyco-DPH in ethanol or POPC were also fitted to unimodal and bimodal continuous lifetime distributions (Table II). X 2 v a l u e s decreased on going from a single exponential (Table I) to a unimodal distributional model (Table II); according to Student's F-test [21], the respective differences in X2 were significant. As already observed for discrete two component fits (Table I), the bimodal distribution patterns (Table I I ) show a predominant long lifetime component and a small fraction centered at short lifetimes. However, X2 values for the biexponential model and the cor-

TABLE I Analysis of the fluorescence emission decay of g l y c o - D P H in ethanol and POPC vesicles', assumingdiscrete exponential componentsb. A

Ethanol 3.59 1.00 4.34 0.94

al

~2

f2

a2

X2

1.00 0.45

m 0.21

_ 0.06

_ 0.55

361.9 9.9

POPC vesictes 5.26

1.00

1.00

--

--

--

6.11

0.91

0.73

1.71

0.09

0.26

93.8

2.2

angles and demodulationsat 10 dflterent modulationfrequencieswere carried out at 20°C. t'The standard deviationsof phase (ore)and modulation(or,,) measurementswere fixedto 0.2° and 0.004, respectively,for the least-squares fittingprocedure (see Theory). "Measurements of phase

192 T A B L E II Analysis of the fluorescence emission decay of glyco-DPH in ethanol and POPC vesicles', assuming continuous Lorentzian lifetime distributions with one or two lifetime-centersb. 'rcellttr, I F W H M I

fl

Teenier.2 FWHM2

/2

X2

Ethanol 3.91 2.38 4.28 0.42

1.00 0.95

-0.003

-0.05

-0.05

51.7 7.1

POPC vesicles 5.53 1.47 6.11 0.05

1.0 0.90

-1.80

-0.11

-0.10

5.8 2.1

11

15

RI

l|

• Measurements of phase angles and demodulations at 10 different modulation frequencies were carried out at 200C. ~Fhe standard deviations of phase (~rp) and modulation (o'm) measurements were fixed to 0.2* and 0.004, respectively, for the least-squares fitting procedure (see Theory).

2 responding bimodal lifetime distribution for glyco-DPH in ethanol are very similar and almost identical for glyco-DPH in POPC. Therefore, on the basis of X2, no judgement can be made as to whether the lifetime properties of glyco-DPH in ethanol or POPC can be better described by either model. Nevertheless, it is interesting that the distribution width of the long lifetime component is significantly broader in ethanol compared with the long label lifetime in POPC (Fig. 6) showing an extremely narrow FWHM that is below the resolution limit [16] of our given data. Provided the distribution model applies to our systems, we suppose, that glyco-DPH may aggregate even in ethanol (and not only in water, see above) at low concentrations thus creating a distribution of different microenvironments around the fluorophor and finally leading to a heterogeneous lifetime distribution. A phospholipid bilayer, on the other hand, may be able to "dissolve" the label homogeneously thus providing free lateral mobility and a very constant environment!for glycoD P H on a nanosecond time-scale. Increasing amounts of sterols in a phospholipid membrane lead to an increase of the glyco-DPH lifetime. This effect is in agreement with the observation that cholesterol condenses pbospholipid bilayers. In its presence, phospholipid packing is tighter and phospholipid acyl chain mobility

i~ki i IIL |

I

I

I

11

i

,

,

I

,

1t

I

I is

I

I III

Fig. 6. Bimodal Lorentzian lifetime distributions of glycoDPH in ethanol (1) and vesicles of palmitoyioleoylphosphatidycholine (POPC) (2) at 20"C.

is reduced [22]. In general, a sterol-phospholipid membrane is a rigid environment favouring longer lifetimes of the exeitated state of fluorophors embedded in the bilayer. DbeaJon Giyco-DPH is an amphipatic derivative of DPH with a very large polar sugar residue which is connected via a polar hydrazyl carbonyi group and two additional methylene groups to the apolar fluorophor. According to its amphiphilicity, it may insert into phospholipid bilayers like a membrane lipid molecule with its main symmetry axis oriented parallel to the phospholipid acyl chains. This is

193

1

2

3

Fig. 7. Tentative model for possibleorientations of DPH (1), TMA-DPH (2) and glyco-DPH (3) in a phospholipid membrane.

shown schematically in a tentative model (Fig. 7) with comparison to the possible membrane orientations of T M A - D P H and DPH. The above indicated possibilities of label orientations are in agreement with results from time-resolved anisotropy studies of D P H and T M A - D P H in bilayers. Whereas D P H motion is best described by two rotational correlation times [23], T M A - D P H clearly behaves as a hindered rotator characterized by a rotational correlation time and limiting anisotropy r®[4,24]. This view is supported by recent results indicating that a considerable proportion of D P H in bilayers is oriented parallel to the membrane surface, whereas T M A - D P H exhibits only a unimodal orientational distribution centered about the bilayer normal [6,19]. Timeresolved anisotropy studies of glyco-DPH are under way to confirm its static and dynamic behaviour in membranes. The fluorophor of glyco-DPH presumably resides deeper in the hydrophobic membrane part compared with TMA-DPH. In the latter molecule, DPH is attached directly to the polar rest, whereas it is separated from the sugar hydroxyl group in glyco-DPH by three carbon atoms. This assumption is consistent with fluorescence anisotropy and lifetime data. Steady state fluorescence anisotropies of glyco-DPH are always lower compared with T M A - D P H in bilayers, According to 2H-NMR [25] and fluorescence anisotropy data [26] membranes exhibit a positive segmental mobility gradient from the surface to the bilayer

center. Furthermore, the predominant long lifetime component of a bimodal T M A - D P H lifetime distribution (~'ccntcr-- 3.94 ns; FWHM = 0.35 ns; unpublished results) is broader compared with the long living glyco-DPH decay distribution (Tcc,t,r = 6.1 ns; FWHM = 0.05 ns). It was suggested that the broad T M A - D P H lifetime distribution originates from a large dielectric gradient due to water penetration or a more viscous environment near the bilayer surface around the fluorophor [17]. This dielectric gradient might be significantly smaller in deeper membrane regions where the fluorophor of glyco-DPH resides thus producing much less lifetime heterogeneity. As a DPH probe for membrane studies glycoD P H has some properties in common with D P H propionyl phosphatidylcholine (DPH-PC) [27, 28]. It contains a DPH propanoyl residue and, as an amphiphile, it should be oriented vertically relative to the membrane surface. In DPH-PC and glyco-DPH, the D P H fluorophor is separated formally by three carbon atoms from the bilayer interface. The localization of the D P H propanoyl group of the phospholipid is defined insofar as it is bound directly to the glycerol backbone of a bilayer constituent and, therefore, linked to the membrane interface; the DPH itself should be forced into the hydrophobic membrane interior. On the other hand, we can only speculate about the localization of the fluorophor of a membranebound glyco-DPH molecule. The hydrazyl group connecting the DPH propanoyl residue with the sugar is very polar and should rather be localized within the hydrophilic membrane surface. Therefore, the DPH fluorophor might be exposed toward the bilayer interface a little bit more compared with the same label in DPH-PC. The fluorescence lifetimes of glyco-DPH (6.1 ns, see Table I) and DPH-PC (6.8 ns) in fluid bilayers of unsaturated [29] or saturated [28] phospholipids are not very different. According to their similar steady-state fluorescence anisotropies (approx. 0.15) in bilayers of palmitoyloleoylphosphatidylcholine or egg phosphatidylcholine at 37°C (own unpublished data), they should experience similar motional freedom; similar anisotropies (approx. 0.15) were also found for DPH-PC in dipal-

194

mitoylphosphatidylcholine above the phase transition temperature [28]. Continuous fluorescence lifetime distribution studies provide, in addition, a very important information on the lateral distribution of glycoDPH within the bilayer surface. In contrast to ethanol, where the label seems to undergo aggregation, glyco-DPH exhibits very narrow lifetime distributions in POPC vesicles (see Fig. 6). Thus the label seems to partition homogeneously over the outer bilayer half, at least in a POPC membrane in the liquid crystalline state. In this respect, glyco-DPH behaves similarly to a fluorescently labeled phospholipid (DPH-PC) which exhibits very narrow lifetime distributions in bilayers [29]. On the other hand glyco-DPH shows an enormous advantage over DPH-PC. It is readily incorporated into artificial as well as into cellular membranes within minutes, whereas effective phospholipid uptake by biological membranes requires the presence of phospholipid transfer proteins [30] and takes much longer time. It is known, on the other hand, that low amounts of glycolipids distribute homogeneously in phosphatidylcholine bilayers above the phase transition temperature [31,32], but show phase separations when the host choline-phospholipid is brought to the gel state [31 ]. Investigations on living cells showed two advantages of the new probe. According to fluorescence microscopy studies, the fluorophor labels selectively the cell surface. Furthermore, it is taken up by cell membranes extremely fast. Uptake is much faster compared with pure phosphatidylcholine bilayers, presumably due to interaction of glyco-DPH with cell surface glycolipids. Whereas it takes tens of minutes for DPH and still some minutes for quantitative incorporation of TMA-DPH in cultured human skin fibroblasts, glyco-DPH insertion is complete within 1 rain (A. Hermetter, B. Rainer, E.N. Ivessa, E. Kaib, J. Loidl, A. Roscher and F. Paltauf, unpublished). This makes, of course, measurements with sensitive living systems extremely effective and reliable. Conclusion

Glyco-DPH is a new membrane label which is amphiphilic but electroneutral, inserts almost in-

stantaneously into artificial and biological membranes, exhibits a defined orientation in a bilayer, and labels specifically the surface membrane of intact cells. It should find wide application in the field of membrane research.

Ackaowle~emems Financial support by the "Fonds zur FiSrderung der wissenschaftlichen Forschung in ()sterreich" (Project No. 5746 B) is gratefully acknowledged.

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