Effect of Ethanol on Membranes: A Fluorescent Probe Study Jane M. Vanderkooi, Ph.D. Using fluorescence techniques to study changes in membrane structure as a function of ethanol concentration and alcoholism, it was found that ethanol primarily affects probes located at the membranewater Interface. Changes In the Upld composition of membranes durlng alcoholism were detected by fluorescent probes that partition into the hydrophobic interior of the membrane.

ECENT work by Chin and Goldstein’ has shown that ethanol in concentrations which are reached physiologically affects the “fluidity” of membranes as detected by spin-label probes. This interesting result deserves further attention since it suggests one mechanism for pharmacologic effects of ethanol. In this article, we discuss the effect of ethanol on membrane structure as monitored by fluorescence probes and compare these effects with those of general anesthetics, and then give a general approach to membrane probe studies. The underlying principle for detection of environmental changes by fluorescent probes is that the excited-state molecule has a different structure than the ground-state molecule and, consequently, can undergo reactions unique to it or those which can be uniquely measured by its luminescence. It is apparent that properties of the excited-state molecule are important for the success of the probe technique. The following are some properties that are useful to select. Long fluorescence lifetime. The interaction of a fluorescent molecule with its environment depends on the time scale of the decay of the excited state; therefore, a molecule with a long fluorescence lifetime will be more likely to be a sensitive probe. High quantum yield. The probe concentration should be low.

R

From the Department of Biochemistry and Biophysics. School of Medicine, University of Pennsylvania, Philadelphia, Pa. Supporied by USPHS Grant GM21699; J.M.V. is supported by Career Development Award GMOO53. Reprint requests should be addressed to Jane M . Vanderkooi, Ph.D.. Department of Biochemistry and Biophysics, School of Medicine, Universiiy of Pennsylvania, Philadelphia. Pa. 19104. 0 1979 by Grune & Stratton. Inc. 01456OO8/79/030140l3601.OO/O

60

Excited-state reactions. Fluorescent molecules that undergo particular excited-state reactions are especially sensitive to environment. Probe molecules that undergo spectral shifts due to solvent rearrangement, protonation, and energy transfer can be selected. Fluorescence polarization measures the mobility of the molecule. In addition,probe location in the membrane is important. Fluorescent probe molecules that are hydrophobic are likely to partition into the hydrocarbon interior of the membrane, whereas amphipathic molecules are more likely to be located at the membrane-water interface. The fluorescent probes chosen for this work and shown in Fig. 1 are anthroyl stearic acid (AS), diphenylhexatriene (DPH), pyrene, and aminopyrene. Each has characteristics that make it sensitive to particular aspects of membrane structure. MATERIALS AND METHODS Pyrene and aminopyrene were obtained from Eastman Chemical Company (Rochester N.Y.); l-phenyld-phenylhexatriene (DPH) was purchased from Aldrich Chemical Company (Milwaukee, Wisc.) and 12-(9-anthroyi)-stearic acid was the gift of Dr. A. Waggoner. Red blood cell ghosts were prepared from normal human blood and from patients who had advanced cirrhosis of the liver, as previously described.* Phospholipid artificial membranes were prepared by sonicating dimyristoyi lecithin (Sigma Chemical Co.. St. Louis. Mo.) with a Bronson sonifier. Steady-state fluorescence was measured with an Hitachi MPF-2A fluorescence spcctrophotometer. D a y of fluorescence was measured on an Ortec photon-counting fluorescent lifetime instrument. RESULTS

Altered Membrane Structure during Alcoholism The fluorescent moiety of anthroyl stearic acid (AS) is located in the hydrocarbon core of the membrane and therefore probes this region. The fluorescence polarization of AS depends on the environment of the probe in the membrane. When cholesterol/phospholipid ratio of red blood cell membrane increases, the fluorescence polarization of AS increases indicating a

Alcoholism: Clinical and Experimental Research, Vol. 3 , No. 1 (January), 1979

FLUORESCENT PROBE STUDY

61

$ I

0-P=o H H ? HF - Y-CH,

NH

0

H C , I

9 9

o=c

H I - C-CH, I

? ? o = c o=c

o=c

Ei Lecithin

Fig. 1.

AS

DPE

ONS

ANS

Fluorescent prober: po8JUon relative to dlmyrlstoyl phosphatldyl chollne (DMPC).

decrease in the fluidity of the membrane (Fig. 2). The result was obtained using red blood cell membranes from patients who had altered cholesterol/phospholipid ratios due to cirrhois of the liver; the same result was obtained using red blood cell membranes in which the normal lipid composition was changed in vitro. This indicates that the lipid composition is the primary reason for the altered membrane fluidity and that fluorescence polarization can be used to detect altered fluidity. We should point out that other fluorescent probes are able to detect changes in membrane structure during liver disease if they fulfill the criteria that they are in the right location and have the right luminescence properties. Additional information can be obtained using pyrene. Pyrene (Fig. l ) , like AS, partitions into the hydrophobic interior of the membrane. It has a long fluorescent lifetime and therefore is likely to undergo excited-state reactions. One such reaction is that oxygen can quench the fluores~ e n c eThe . ~ amount of quenching depends on the oxygen present and the diffusion coefficient of

704 0

0.4

08

1.2

1.6

2.0

-

2.4

Free Cholesterol/Phospholipid (mol /mol 1 Fig. 2. Effect of chole8teroCphosphollpld mole retlos on fluorescence anisotropy of AS. The sample contained 0.154 M NaCI, 10-30 MAS, and 0.2-0.4 mg protein/ml of (0) normal red blood cell gho8tr; ( 0 .A) red blood cells from two patlent8 with spur cells; end (0, m, A) normal cell8 In whlch the cholesterol reti08 were altered by Incubating with cholesterol. Data are from Vanderkool at el.*Fluorescence anlsotropy was taken on a Perkln-Elmer YPF2A fluorimeter using 380 nm and 440 nm as excitation and emission wavelengths.

JANE

62

RBC

DPL

-

1

0.003

Fig. 3.

0.0032

O.CO34

0.rn

0.0031

00033

0.0035

Fiuorosconcointonrlty of pyreno in mombranosas

a function of tomporature. All samples containod 0.6 p Y pyreno and 0.1 mYPO, (pH 7.4). Tho dimyristoyl phoaphatldyi choiino (DML) and dipalmltoyl phosphatldyl choiino (DPL) aamples containod 3.3 mg iocithin/ml. Tho umpio Iabelod DML/CHOL containod 0.07 mg choiosteroi and 0.23 mg dimyristoyl phosphatldyl choiino/ml. Erythrocyte ghost preparations (RBC) contained 0.3 mg protoin/mi. Opon symbols indC cote meawroments mado ascending in tomporaturo; solid symbols dorcending. F, is tho rolstlvo flUOrOSCOnC0 tomporz~ turo in the u m p k oquiiibratod with nitrogen, and F Is the rolstlvo fluororconce intonrlty in an idontical umplo oquiiC brated with oxygon. Data are from Fischkoff and Vanderkooi, 1975.'

oxygen. Therefore, the degree of quenching can be used to calculate the oxygen-diffusion coefficient. Figure 3 shows the quenching of pyrene fluorescence by oxygen as a function of temperature for various artificial and natural membranes. At the phase transition of artificial membranes there is a large increase in the diffusion rate of oxygen, as indicated by the change in the fluorescence yield. Data such as these reveal that the presence of cholesterol in the membrane decreases the oxygen-diffusion rate.

M. VANDERKOOI

Direct Alcohol Interaction with Membranes The possibility exists that ethanol affects the membrane fluidity directly. The fluorescent probes used are diphenyl hexatriene, located in the membrane interior, and aminopyrene, located on the surface (Fig. 1). The rotational relaxation of DPH, obtained from fluorescence polarization measurement, is shown in Fig. 4. Increasing concentrations of commonly used anesthetics and ethanol shift the phase transition of dimyristoyl phosphatidyl choline vesicles to lower temperatures. One difference between the anesthetics and ethanol is that the concentration required for ethanol to shift the phase transition by 2OC is more than two orders of magnitude greater than for the general anesthetics? In fact, it is much higher than is physiologically obtained. These data, taken by themselves, would indicate that alcohol has little effect on the hydrocarbon interior of the membrane at physiologic concentrations. Aminopyrene (Fig. 1) is an interesting fluorescent probe because it is located at the membrane-water interface. In addition, the amino group is attached to the ring, therefore, its fluorescence properties are affected by protonation of this moiety. Figure 5 shows that addition of 1 mM ethanol resulted in a 5% decrease in fluorescence intensity, and there was a further decrease when more alcohol was added. The reason alcohol affects the fluorescence of aminopyrene is not yet understood. It could be due in part to excited-state protonation reactions. It would seem, however, that alcohol has affected the structure of the interface (the structured water or choline head groups), which is reflected in altered fluorescence properties of aminopyrene. This is a hypothesis that could be studied in detail using nuclear magnetic relaxation techniques. SUMMARY

Altered cholesterol/phospholipid ratios in membranes of the chronic alcoholic result in changed membrane fluidity, as detected by fluorescent probes. At physiologic concentrations, there is little effect of alcohol on the hydrocarbon core of the membrane; however, a fluorescent probe located at the interface is affected by low concentrations of ethanol.

FLUORESCENT PROBE STUDY

63

G Halothane

I

I

I

I

.003i

,0033

I

I

I

,0035

1

.0032

1

,0034

‘ .od36

.Od=

-

.od34

I/T

,0036 M Y - 465

Fig. 4. Effect of anesthetics on the rotational correlation time of DPH in dimyristoyl phosphatidyi choline vesicles. The sample contained 0.1 mg dimyristoyi phorphatidyi choiine/mi, 0.05 M NH, CH ,O ,, (pH 7.0) and 0.2 M DPH. Trliene: 0 ,no addition; A, 5.4 mM; e, 10.0 mM. Halothane: 0. no addition; 0 , 14 mM; A, 21 mM; e, 44 mM. Ethanol: 0 , no addltiona; A, 1.8 Methanol; e, 3.6 Methanol. Trliene (trichioroethyiene) was obtained from Ayerst laboratories Inc. (New York. N.Y.). Thymoi-free “halothane” (2-bromo2chloro-l,l,l, trlfluoroethane) was a gift of L A . Small (ICI United States. inc., Dighton, Mass.). Data are from Vanderkooi et a.i’

ACKNOWLEDGMENTS I

OH61

Exc’’o‘lon

The author wishes to thank Drs. Jane Chin and Dora Goldstein for the suggestion that ethanol would primarily affect probes located at the membrane-water interface.

REFERENCES

- I

280 Mo

3 b Unml

4bO A h 1

Fig. 5. Aminopyrene fluorescence in dimYrlstoyl phorphb tldyi choline vesicles. (Left) Excitation spectra of 1 b~Maminopyrene in 0.5 mg dimyristoyi phosphatidyl choline and 10 mM phosphate at the pH indicated. Emiasion wavelength at 440 nm. (Right) Emission spectra of the ram. sample at pH 4.0 before (a) and after (b) addition of 1 mMethanol.

I . Chin JH, Goldstein DG: Drug tolerance in biomembranes: A spin label study of the effects of ethanol. Science 196:684-685,I977 2. Vanderkooi JM, Fischkoff S, Chance B. Cooper R: Fluorescent probe analysis of the lipid architecture of natural and experimental cholesterol-rich membranes. Biochemistry 13:l589-l594..1974 3. Fischkoff S, Vanderkooi JM: Oxygen diffusion in biological and artificial membranes determined by the flue rochrome pyrene. J Gen Physiol 65:663-676, 1975 4. Vanderkooi JM. Landesburg R. Selick H, McDonald GG: Interaction of general anesthetics with phospholipid vesicles and biological membranes. Biochim Biophys Acta 464~1-16.1977