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A time-resolved fluorescence anisotropy study of bilayer membranes containing α-tocopherol
Vo1.158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 386-391
Janua~ 31,1989
A TIME-RESOLVED FLUORESCENCE ANISOTROPY STUDY OF BILAYER MEMBRANES CONTAINING ot-TOCOPHEROL
Roger H. Bisby1 and David J.S.Birch 2
1Department of Biological Sciences, University of Salford, Salford M5 4WT, UK 2Department of Physics and Applied Physics, University of Strathclyde, Glasgow G4 ONG, UK Received December 6, 1988
Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing c~-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing c~-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly. ~ 1989 AcademicPress, Znc.
The major role of c,-tocopherol in cellular membranes is to act as an antioxidant (1,2), although an additional function in the stabilization of membranes has been proposed (3,4).
As an antioxi-
dant, c~-tocopherol donates a hydrogen atom to a lipid peroxyl radical, terminating the peroxidative free radical chain. The resulting tocopherol radical is repaired by ascorbate in the aqueous compartment. The overall reaction might be facilitated either by displacement of the polar peroxyl radical to the membrane interface (5), by a radical "relay mechanism" in the phytyl chain (6), or by diffusional mobility of the a-tocopherol in the bilayer (7). a-tocopherol in a membrane is very slow (8).
Recent evidence shows that "flip-flop" of
Previous steady-state fluorescence (9-14) and 2H
NMR (15) experiments suggest that a-tocopherol and tocopherol acetates cause a decrease in 'fluidity" of bilayer membranes above the gel to liquid crystalline phase transition temperature. In contrast,
lsC NMR (16) and spin-label (17) studies have been interpreted to indicate either an increase
in 'fluidity' or an insignificant effect respectively. In this study we have used a diphenylhexatrienephosphatidylcholine conjugate (DPH-PC) and time-resolved fluorescence anisotropy measurements to provide more detail of the change in membrane 'fluidity' caused by a-tocopherol. MATERIALS AND METHODS (+)-ct-Tocopherol (natural, >99%) was obtained from Fluka. L-ct -Dipalmitoylphosphatidylcholine (DPPC, synthetic, >99%) was purchased from Sigma. DPH-PC was synthesised by esterification of egg yolk lysophosphatidylcholine in the 2-position with propionyl-DPH (18) and was a gift from Drs E.W.Thomas and C.G.Morgan (University of Salford). Unilamellar vesicles (ULV's) of DPPC containing c~-tocopherol were obtained by the ethanol injection method (19). The concentration of c~-tocopherol was determined by spectrophotometry, taking ~(295nm)=3000 M-lcm -1 in ethanol (20). DPH-PC was added to the ethanol solutions prior to Abbreviations used:- DPH, diphenylhexatriene; DPPC, dipalmitoylphosphatidylcholine; ULV, unilamellar vesicle. 00o6-291x/89 $1.50 Copyright © 1989 by Academic Press, Inc. All riehts of reoroduction in any form reserved.
386
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
injection to give a probe:lipid ratio of 1:200. The concentration of DPH was determined taking ~(353nm)=6.104 M-lcm-1. Time-resolved fluorescence measurements were made using multiplexed time-correlated single photon counting with an Edinburgh Instruments Model 299T fluorometer, used in an L-format with a rotating emission polarizer, and with simultaneous acquisition of the fluorescence and excitation decay profiles (SAFE) (21). This arrangement corrects automatically for both temporal and intensity fluctuations of the hydrogen spark source.
RESULTS AND DISCUSSION DPH-PC was used as a fluorescence probe to evaluate the effect of increasing a-tocopherol content of DPPC bilayers at 47 C, above the phase transition temperature in these membranes. The (~-tocopherol content was varied up to 36
mole%, which is close to the maximum of 40 mole%
which can be accomodated in such bilayers (20). The decay of the total fluorescence of DPH-PC, evaluated from the sum of the polarized components, in DPPC ULV's alone was found to be best fitted by a double exponential function with lifetimes (and intensities) of 6.46 ns (88%) and 2.88 ns (12%), as has been found previously (22,23). Addition of ~-tocopherol to the membranes led to a decrease of both decay times and an increase in the relative intensity of the shorter lifetime component as shown in Figure 1.
The decrease in fluorescence lifetimes may be due to an increase in
polarity experienced by the DPH moiety, since it is known that the DPH lifetime decreases with increasing solvent polarity (24).
The increase in polarity may be due to exposure of the fluorophore,
expected to be located in the hydrocarbon region of the bilayer for DPH-PC, to the hydrophilic chromanol ring of a-tocopherol.
The fluorescence lifetime of pyrene in bilayer membranes has also
been reported to be quenched by a-tocopherol (14), but tocopherol acetate has no such effect (9). Previous determinations of the effect of a-tocopherol on the 'fluidity' of bilayer membranes using depolarization of DPH fluorescence have relied on steady-state measurements (11-13). Since the fluorescence lifetime appears in the Perrin equation, the above results show that without corresponding lifetime measurements, steady-state data are open to misinterpretation. Time resolved measurements
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FIGURE 1. Effect of a-tocopherol content on the fluorescence decay of DPH-PC in DPPC ULV's at 47 C in phosphate buffer (20 mM, pH 7.3). The data were fitted to double exponential decay function with lifetimes r 1 (o) and r 2 (o). The percentage of total intensity for the short lifetime component ( i ) is also shown. 387
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F I G U R E 2. D e c a y profiles f o r D P H - P C in D P P C U L V ' s at 47 C c o n t a i n i n g 13.1 mole% a - t o c o pherol. A : - F l u o r e s c e n c e decays w i t h parallel ( I v ) a n d p e r p e n d i c u l a r ( I n ) polarisers. T h e channel w i d t h is 102ps a n d the f l u o r e s c e n c e decays w e r e m e a s u r e d s i m u l t a n e o u s l y w i t h the l a m p pulse in 13 hours. B : - A n i s o t r o p y decay. Fitting to I v - I H using impulse r e c o n v o l u t i o n g a v e a rotational c o r r e l a tion time ¢=2.18 + 0.08 ns a n d a residual a n i s o t r o p y rlnr =0.095 -+ 0.001 w i t h a n o r m a l i s e d c h i s q u a r e d o f 1.07.
388
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were therefore undertaken of the decay of DPH-PC fluorescence anisotropy in DPPC bilayers containing c~-tocopherol. The tlme-resolved decays of the parallel (Iv) and perpendicular (Ia) components of DPH-PC fluorescence in DPPC ULV's at 47 C containing 13 mole% c~-tocopherol are shown in Figure 2A and the corresponding decay of the fluorescence anisotropy (r(t)) is depicted in Figure 2B. The data are clearly indicative of restricted rotational mobility of the probe in the membrahe (23,25). We have adopted a simple wobbling-in-cone analysis (26) to interpret this data (equations 1 and 2), although eventually a more complex analysis (17) may prove to be more appropriate. Using an impulse reconvolution procedure with two fluorescence decay components, the anisotropy data was fitted to equation 1. For a molecule such as DPH in which the absorption and emission dipoles coincide with the long molecular axis, this gives a value of rinf which is related (26,28) to the half-cone angle (0e) within which restricted diffusion occurs and the second rank order parameter (S) through equation 2. The diffusion constant for wobbling in the cone (Dw) is related to the apparent rotational relaxation time (~) in equation 1 by ~=a/D w (26,28). r(t)= (ro-rinf)exp [- ~] + r M
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F I G U R E 3. Effect of a-tocopherol content on parameters evaluated from the decay of fluorescence anisotropy for DPH-PC in DPPC ULV's at 47 C in phosphate buffer (20 raM, pH 7.3), analysed according to the wobbling-in-cone model. A:- Order parameter (S); B:- half-cone angle (0~); C:- wobbling diffusion constant (Dw), units 10-2 ns-t.
389
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Values of a were calculated from the expression given by Lipari and Szabo (28). Using this analysis, the effect of a-tocopherol on S, Dw and 0c for DPH-PC in DPPC ULV's at 47 C are shown in Figure 3. The data clearly show that there is a progressive decrease in the halfcone angle (0e) with increasing a-tocopherol content of the bilayer, and that the order parameter (S) for the bilayer reported by DPH-PC progressively increases. The changes in S and 0e are most apparent on addition of between 5 and 20 mole% a-tocopherol to the bilayer. The order parameter reported by DPH-PC in fluid phase DPPC alone (0.41) is greater than that indicated by DPH (S=0.3) (29), as would be expected from the way in which the fluorophore in DPH-PC is anchored at one extremity (23). The wobbling diffusion constant (Dw) is determined from both rinf and ~ and is therefore subject to larger error. The value of Dw appears to be unaffected by a-tocopherol, except perhaps at the highest concentration of a-tocopherol studied (36.3 mole%) when there is an indication of a small increase. The effect of a-tocopherol on membrane "fluidity" has been likened to that of cholesterol (11, 15). The fluorescence lifetime of DPH in ULV's of dimyristroyl-PC above the phase transition temperature increases slightly with increasing cholesterol content of the membrane (30), in contrast to the decrease in DPH-PC fluorescent lifetime on addition of ~-tocopherol. The effect of cholesterol on the restricted rotational diffusion of DPH above the phase transition temperature is similar to that of a-tocopherol, with a marked decrease in the half-cone angle for wobbling, but with no significant change in Dw, except at the highest content of cholesterol (40 mole%) examined by Kinosita and Ikegami (30), when a small decrease was measured. Time-resolved fluorescence anisotropy measurements with DPH-PC as a probe therefore show that ~-tocopherol increases the order parameter in bilayers of DPPC at a temperature above that of the phase transition. Further work is underway using the fluorescence of c~-tocopherol itself as a probe of membrane dynamics.
ACKNOWLEDGMENTS. DJSB aknowledges the support of SERC and Edinburgh Instruments Ltd., in addition to Nuffield Foundation and Royal Society/SERC Fellowships.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Scott,M.L. (1980) Fed.Proc. 39, 2736-2739. McCay,P.B. (1985) Ann.Rev.Nutr. 5, 323-340. Diplock,A.T. and Lucy,J.A. (1973) FEBS Lett. 29, 205-210. Diplock,A.T. (1983) in CIBA Symposium No.101 - The Biology of Vitamin E, pp.45-55. Pitman, London. Barclay,L.R.C. and Ingold,K.U. (1981) J.Am.Chem.Soc. 103,6478-6485. Ivanov,I.I. (1985) J.Free Rad.Biol.Med. 1, 247-253. Ahmed,S. and Bisby,R.H. (1989) Free Rad.Biol.Med., In press. Tyurin,V.A., Kagan,V.E., Serbinova,E.A., Gorbunov,N.V., Erin,A.N., Prilipko,L.L. and Stoichev,Ts.S. (1986) Byull.Eksp.Biol.Med. 102, 689-692. Schmidt,D., Steffen,H. and Von Planta,C. (1976) Biochim.Biophys.Acta 443, 1-9. Yamauehi,R. and Matsushita,S. (1979) Agric.Biol.Chem. 43,357-362. Fukuzawa,K., Chida,H. and Suzuki,A. (1980) J.Nutr.Sci.Vitaminol. 26, 427-434. Steiner,M. (1981) Biochim.Biophys.Acta 640, 100-105. Massey,J.B., She,H.S. and Pownall,H.J. (1982) Biochem.Biophys.Res.Commun. 106,842-847. Ohyashiki,T., Ushiro,H. and Mohri,T. (1986) Biochim.Biophys.Acta 858, 294-300. Wassall.S.R., Thewalt,J.L., Wong,L., Gorrissen,H. and Cushley,R.J. (1986) Biochemistry 25, 319-326. Cushley,R.J. and Forrest,B.J. (1977) Can.J.Chem. 55, 220-226. Cushley,R.J., Forrest,B.J., Gillis,A. and Tribe,J. (1979) Can.J.Chem. 57, 458-465. 390
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18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Morgan,C.G., Thomas,E.W., Moras,T.S. and Yianni,Y.P. (1982) Biochim.Biophys.Acta 692, 196-201. Batzri,S. and Korn,E.D. (1973) Biochim.Biophys.Acta 298, 1015-1019. Villalain,J., Aranda,F.J. and Gomez-Fernadez,J.C. (1986) Eur.J.Biochem. 158, 141-147. Birch,D.J.S., Holmes,A.S., Gilchrist,J.R., Imhof,R.E. and Nadolski,B. (1987) J.Phys.E:Sci.Instrum. 20, 471-473. Cranney,M., Cundall,R.B., Jones,G.R., Richards,J.T. and Thomas,E.W. (1983) Biochim.Biophys.Acta 735, 418-425. Parenta,R.A. and Lentz,B.R. (1985) Biochemistry 24, 6178-6185. Cehelnik,E.D., Cundall,R.B., Lockwood,J.R. and Palmer,T.F. (1975) J.Phys.Chem. 79, 1369-1376 Dale,R.E, Chen,L.A. and Brand,L. (1977) J.Biol.Chem. 252, 7500-7510. Kinosita,K, Ikegami,A. and Kawato,S. (1982) Biophys.J. 37, 461-464. Zannoni,C. Arcioni,A. and Cavatorta,P. (1983) Chem.Phys.Lipids 32, 179-250. Lipari,G. and Szabo,A. (1980) Biophys.J. 30, 489-506. Heyn,M.P. (1979) FEBS Lett. 108, 359-364. Kinosita,K. and Ikegami,A. (1984) Biochim.Biophys.Acta 769, 523-527.
391
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 386-391
Janua~ 31,1989
A TIME-RESOLVED FLUORESCENCE ANISOTROPY STUDY OF BILAYER MEMBRANES CONTAINING ot-TOCOPHEROL
Roger H. Bisby1 and David J.S.Birch 2
1Department of Biological Sciences, University of Salford, Salford M5 4WT, UK 2Department of Physics and Applied Physics, University of Strathclyde, Glasgow G4 ONG, UK Received December 6, 1988
Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing c~-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing c~-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly. ~ 1989 AcademicPress, Znc.
The major role of c,-tocopherol in cellular membranes is to act as an antioxidant (1,2), although an additional function in the stabilization of membranes has been proposed (3,4).
As an antioxi-
dant, c~-tocopherol donates a hydrogen atom to a lipid peroxyl radical, terminating the peroxidative free radical chain. The resulting tocopherol radical is repaired by ascorbate in the aqueous compartment. The overall reaction might be facilitated either by displacement of the polar peroxyl radical to the membrane interface (5), by a radical "relay mechanism" in the phytyl chain (6), or by diffusional mobility of the a-tocopherol in the bilayer (7). a-tocopherol in a membrane is very slow (8).
Recent evidence shows that "flip-flop" of
Previous steady-state fluorescence (9-14) and 2H
NMR (15) experiments suggest that a-tocopherol and tocopherol acetates cause a decrease in 'fluidity" of bilayer membranes above the gel to liquid crystalline phase transition temperature. In contrast,
lsC NMR (16) and spin-label (17) studies have been interpreted to indicate either an increase
in 'fluidity' or an insignificant effect respectively. In this study we have used a diphenylhexatrienephosphatidylcholine conjugate (DPH-PC) and time-resolved fluorescence anisotropy measurements to provide more detail of the change in membrane 'fluidity' caused by a-tocopherol. MATERIALS AND METHODS (+)-ct-Tocopherol (natural, >99%) was obtained from Fluka. L-ct -Dipalmitoylphosphatidylcholine (DPPC, synthetic, >99%) was purchased from Sigma. DPH-PC was synthesised by esterification of egg yolk lysophosphatidylcholine in the 2-position with propionyl-DPH (18) and was a gift from Drs E.W.Thomas and C.G.Morgan (University of Salford). Unilamellar vesicles (ULV's) of DPPC containing c~-tocopherol were obtained by the ethanol injection method (19). The concentration of c~-tocopherol was determined by spectrophotometry, taking ~(295nm)=3000 M-lcm -1 in ethanol (20). DPH-PC was added to the ethanol solutions prior to Abbreviations used:- DPH, diphenylhexatriene; DPPC, dipalmitoylphosphatidylcholine; ULV, unilamellar vesicle. 00o6-291x/89 $1.50 Copyright © 1989 by Academic Press, Inc. All riehts of reoroduction in any form reserved.
386
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
injection to give a probe:lipid ratio of 1:200. The concentration of DPH was determined taking ~(353nm)=6.104 M-lcm-1. Time-resolved fluorescence measurements were made using multiplexed time-correlated single photon counting with an Edinburgh Instruments Model 299T fluorometer, used in an L-format with a rotating emission polarizer, and with simultaneous acquisition of the fluorescence and excitation decay profiles (SAFE) (21). This arrangement corrects automatically for both temporal and intensity fluctuations of the hydrogen spark source.
RESULTS AND DISCUSSION DPH-PC was used as a fluorescence probe to evaluate the effect of increasing a-tocopherol content of DPPC bilayers at 47 C, above the phase transition temperature in these membranes. The (~-tocopherol content was varied up to 36
mole%, which is close to the maximum of 40 mole%
which can be accomodated in such bilayers (20). The decay of the total fluorescence of DPH-PC, evaluated from the sum of the polarized components, in DPPC ULV's alone was found to be best fitted by a double exponential function with lifetimes (and intensities) of 6.46 ns (88%) and 2.88 ns (12%), as has been found previously (22,23). Addition of ~-tocopherol to the membranes led to a decrease of both decay times and an increase in the relative intensity of the shorter lifetime component as shown in Figure 1.
The decrease in fluorescence lifetimes may be due to an increase in
polarity experienced by the DPH moiety, since it is known that the DPH lifetime decreases with increasing solvent polarity (24).
The increase in polarity may be due to exposure of the fluorophore,
expected to be located in the hydrocarbon region of the bilayer for DPH-PC, to the hydrophilic chromanol ring of a-tocopherol.
The fluorescence lifetime of pyrene in bilayer membranes has also
been reported to be quenched by a-tocopherol (14), but tocopherol acetate has no such effect (9). Previous determinations of the effect of a-tocopherol on the 'fluidity' of bilayer membranes using depolarization of DPH fluorescence have relied on steady-state measurements (11-13). Since the fluorescence lifetime appears in the Perrin equation, the above results show that without corresponding lifetime measurements, steady-state data are open to misinterpretation. Time resolved measurements
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FIGURE 1. Effect of a-tocopherol content on the fluorescence decay of DPH-PC in DPPC ULV's at 47 C in phosphate buffer (20 mM, pH 7.3). The data were fitted to double exponential decay function with lifetimes r 1 (o) and r 2 (o). The percentage of total intensity for the short lifetime component ( i ) is also shown. 387
Vol.
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1989
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F I G U R E 2. D e c a y profiles f o r D P H - P C in D P P C U L V ' s at 47 C c o n t a i n i n g 13.1 mole% a - t o c o pherol. A : - F l u o r e s c e n c e decays w i t h parallel ( I v ) a n d p e r p e n d i c u l a r ( I n ) polarisers. T h e channel w i d t h is 102ps a n d the f l u o r e s c e n c e decays w e r e m e a s u r e d s i m u l t a n e o u s l y w i t h the l a m p pulse in 13 hours. B : - A n i s o t r o p y decay. Fitting to I v - I H using impulse r e c o n v o l u t i o n g a v e a rotational c o r r e l a tion time ¢=2.18 + 0.08 ns a n d a residual a n i s o t r o p y rlnr =0.095 -+ 0.001 w i t h a n o r m a l i s e d c h i s q u a r e d o f 1.07.
388
Vol. 158, No. 2, 1989
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
were therefore undertaken of the decay of DPH-PC fluorescence anisotropy in DPPC bilayers containing c~-tocopherol. The tlme-resolved decays of the parallel (Iv) and perpendicular (Ia) components of DPH-PC fluorescence in DPPC ULV's at 47 C containing 13 mole% c~-tocopherol are shown in Figure 2A and the corresponding decay of the fluorescence anisotropy (r(t)) is depicted in Figure 2B. The data are clearly indicative of restricted rotational mobility of the probe in the membrahe (23,25). We have adopted a simple wobbling-in-cone analysis (26) to interpret this data (equations 1 and 2), although eventually a more complex analysis (17) may prove to be more appropriate. Using an impulse reconvolution procedure with two fluorescence decay components, the anisotropy data was fitted to equation 1. For a molecule such as DPH in which the absorption and emission dipoles coincide with the long molecular axis, this gives a value of rinf which is related (26,28) to the half-cone angle (0e) within which restricted diffusion occurs and the second rank order parameter (S) through equation 2. The diffusion constant for wobbling in the cone (Dw) is related to the apparent rotational relaxation time (~) in equation 1 by ~=a/D w (26,28). r(t)= (ro-rinf)exp [- ~] + r M
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389
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Values of a were calculated from the expression given by Lipari and Szabo (28). Using this analysis, the effect of a-tocopherol on S, Dw and 0c for DPH-PC in DPPC ULV's at 47 C are shown in Figure 3. The data clearly show that there is a progressive decrease in the halfcone angle (0e) with increasing a-tocopherol content of the bilayer, and that the order parameter (S) for the bilayer reported by DPH-PC progressively increases. The changes in S and 0e are most apparent on addition of between 5 and 20 mole% a-tocopherol to the bilayer. The order parameter reported by DPH-PC in fluid phase DPPC alone (0.41) is greater than that indicated by DPH (S=0.3) (29), as would be expected from the way in which the fluorophore in DPH-PC is anchored at one extremity (23). The wobbling diffusion constant (Dw) is determined from both rinf and ~ and is therefore subject to larger error. The value of Dw appears to be unaffected by a-tocopherol, except perhaps at the highest concentration of a-tocopherol studied (36.3 mole%) when there is an indication of a small increase. The effect of a-tocopherol on membrane "fluidity" has been likened to that of cholesterol (11, 15). The fluorescence lifetime of DPH in ULV's of dimyristroyl-PC above the phase transition temperature increases slightly with increasing cholesterol content of the membrane (30), in contrast to the decrease in DPH-PC fluorescent lifetime on addition of ~-tocopherol. The effect of cholesterol on the restricted rotational diffusion of DPH above the phase transition temperature is similar to that of a-tocopherol, with a marked decrease in the half-cone angle for wobbling, but with no significant change in Dw, except at the highest content of cholesterol (40 mole%) examined by Kinosita and Ikegami (30), when a small decrease was measured. Time-resolved fluorescence anisotropy measurements with DPH-PC as a probe therefore show that ~-tocopherol increases the order parameter in bilayers of DPPC at a temperature above that of the phase transition. Further work is underway using the fluorescence of c~-tocopherol itself as a probe of membrane dynamics.
ACKNOWLEDGMENTS. DJSB aknowledges the support of SERC and Edinburgh Instruments Ltd., in addition to Nuffield Foundation and Royal Society/SERC Fellowships.
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