Chemistry and Physics of Lipids, 63 (1992) 69-75 Elsevier Scientific publishers Ireland Ltd.
69
Location and dynamics of a-tocopherol in model phospholipid membranes with different charges Kenji Fukuzawa a, Wataru Ikebata a, Akira Shibata a, Itsumaro Kumadaki b, Tatsumi Sakanaka c and Shiro Urano c aFaculty of Pharmaceutical Sciences, Tokushima University, 1-78 Shomachi, Tokushima 770, bFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 and CTokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, ltabashi-ku, Tokyo 173 (Japan)
(Received June 11th, 1992; revision received September 11th, 1992; accepted October 6th, 1992) Studies were made on the position and dynamics of the OH-group of a-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (Tl) relaxation times at the 5a-position of a-tocopherol labeled with 13C-or CIgF3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 tool% ~t-tocopherol, though incorporation of 10 mol% ascorbylpalmitate decreased the zetapotentials of EYPC and EYPC-SA liposomes. The ~ O stretching band (1235 cm-I) of the phosphate group and C-m-Ostretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transforminfrared (FT-IR) spectroscopy, were not changed by incorporation of a-tocopherol. These results suggest that no specific interaction occurred between the OH-group of a-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearicacids (n-NSs) on the intrinsic fluorescence of a-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of a-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface. No difference was found in the oxidation rates of a-tocopherol induced by water-sohible 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) in gel state dipalmitoylphosphatidylcholine (DPPC) liposomes and liquid crystalline state DMPC liposomes, indicating that the OH-group is not located deep in the hydrophobic region. Key words: a-tocopherol; vitamin E; liposomes; spin probe; antioxidant
Introduction ct-Tocopherol (vitamin E) is an important component o f biological membranes and is generally considered to protect the unsaturated fatty acyl Correspondence to: K. Fukuzawa, Faculty of Pharmaceutical Sciences, Tokushima University, 1-78 Shomachi, Tokushima 770, Japan. Abbreviations: AAPH, 2,2 '-azobis(2-amidinopropane)dihydrochloride; DCP, dicetylphosphate; DMPC, diniyristoylphospbatidylcholine; DPPC, dipalmitoylphosphatidylcholine; EYPC, egg yolk phosphatidylcholine; FT-IR, Fourier transforminfrared; HEPES, N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid; NMR, nuclear magnetic resonance; n-NS, n-(Noxyl-4,4-dimethyloxasolidin-2-yl)stearic acid; SA, stearylamine.
chains o f m e m b r a n e lipids from peroxidation [1,2]. It is an amphiphilic molecule consisting o f two functional domains, a c h r o m a n o l nucleus with an O H - g r o u p and a h y d r o p h o b i c phytyl side chain. There is evidence that the polar O H - g r o u p terminates the chain o f peroxidation reactions by trapping free radicals (mainly peroxyl radicals) [3,4], whereas the h y d r o p h o b i c phytyl side chain associates with fatty acid and retains ot-tocopherol in the m e m b r a n e bilayer to stabilize the m e m b r a n e and enhance the effectiveness o f the antioxidant action o f ot-tocopherol [5-7]. There are reports that the O H - g r o u p o f o~tocopherol is located at the m e m b r a n e surface [8-11] or in the m e m b r a n e close to the surface
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 [12-15]. Proposals have been made, moreover, that the OH-group hydrogen bonds phosphate [9,10] or carbonyl [14,15] groups of adjacent phospholipids. On the other hand, recent fluorimetric studies demonstrated that the chromanol nucleus of o~-tocopherol is located in the hydrophobic inner region of fatty acyl chains in bilayer membranes in a medium of relatively high ionic strength [16,17] and in monolayer micenar membranes [18]. Here we report studies on the position of the OH-group of ot-tocopherol in phospholipid vesicles using various physical techniques and discuss the dynamics of radical scavenging by txtocopherol in phospholipid membranes. Materials and Methods
Materials o~-Tocopherol (over 99% pure) was supplied by Eisai Pharmaceutical Co., Tokyo. 5-, 7-, 12- and 16(N-oxyl-4,4'-dimethyloxazolidin-2-yl)stearic acids (5-NS, 7-NS, 12-NS and 16-NS) were obtained from Aldrich Chemical Co., Milwaukee, WI. 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and stearylamine (SA) were from Wako Pure Chemical Industries. N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), dimyristoylphosphatidylcholine (DMPC), dicetylphosphate (DCP) and dipalmitoylphosphatidylcholine (DPPC) were purchased from Sigma Chemical Co., St. Louis, MO. Egg yolk phosphatidylcholine (EYPC) was obtained from Nippon Oil and Fats Co., Ibaraki. All other reagents were of analytical grade. Preparations of a-tocopherol labeled with 13C or 19F at the 5a-position were synthesized as described previously [19,20].
Preparation of liposomes Small unilamellar vesicles (SUV) were prepared as described previously [21] with minor modifications and used for experiments other than the NMR study. Stock solutions of EYPC or DMPC in chloroform with or without SA or DCP were evaporated to dryness under nitrogen in a roundbottom flask. The thin lipid film on the glass wall was dispersed with a Vortex mixer in l0 mM HEPES buffer (pH 7.0) to obtain multilamellar
vesicles (MLV). These MLV were disrupted to SUV by sonication in a Bransonic-12 sonic bath at 40°C. Spin probes were incorporated into the liposomal membranes as reported by Takahashi et al. [11] by adding a solution of SUN containing tx-tocopherol to a thin film of n-NS in a flask and sonicating the mixture at 40°C. Single bilayer liposomes of uniform size and high concentration were required to obtain high resolution NMR spectra. We prepared SUV of about 110 nm diameter as reported previously [6]. A volume of 1 ml of ethanol containing 20 /zraol of EYPC, 1.5/~mol of 13C- or 19F-tx-tocopherol and 1 pmol of DCP or SA was rapidly injected through a Hamilton syringe into 20 ml of Tris-HCl buffer (pH 7.4). The suspension was concentrated to 0.5 mi by Amicon ultrafiltration using an XM-100A membrane with rapid stirring under nitrogen at a pressure of 2 atm/cm 2. The opalescent suspension obtained was applied to a Sepharose 4B column and material was eluted with Tris-HC1 (pH 7.4). Finally 0.5 ml of single bilayer liposome suspension was obtained by combining the liposome fractions and concentrating them in dialysis tubing against Ficol 400 (Pharmacia).
Measurements of 13C- and 19F-spin-lattice relaxation times 13C- and 19F-NMR spectra were recorded at 20°C in a Varian 400 MHz spectrometer using D20 as solvent and sodium 2,2-dimethyl-2silapentane-5-sulfonate as internal standard. Spinlattice (Tl) relaxation times were measured by the inversion recovery technique of Void et al. [22]
Measurement of FT-IR spectra A Perkin Elmer model 1720 FT-IR spectrophotometer was used. The samples were analyzed in a Circle Cell (a cylindrical attenuated total reflectance cell). Experiments were carded out in a frequency range of 800-3200 cra -t, with a resolution of 2 cm -I and data were Fourier transformed and averaged after 30 scans.
Determination of zeta-potential The zeta-potential of liposomes was measured in a LAZER ZEE model 501 apparatus (PEN K E N Co., USA).
71
Oxidation of ~-tocopherol by AAPH DMPC or DPPC liposomes containing ~,-tocopherol were incubated with AAPH at 30°C. The reaction was started by the addition of AAPH. The amount of o~-tocopherol oxidized was measured at room temperature by monitoring decrease of fluorescence intensity (Ex: 297 nm, Era: 326) in hexane after extraction of the ¢x-tocopberol with 5 volumes of EtOH-bexane (1:5, v/v). Results
Results of NMR [6,8-10], electron spin resonance (ESR) [7,11], FT-IR [23] and differential scanning calorimetric [23,24] studies and with fluorescent probes [13,16] have indicated that the chromanol ring of ¢x-tocopherol is located near the polar moiety of the lipid matrix. However, these studies did not show whether polar interaction occurs between the chromanol OH-group of ottocopberol and the phosphate or ester carbonyl group of the phospholipid. Figure 1 shows the FT-IR spectra of DMPC liposomes with and without o~-tocopherol at 37°C. The strong bands at 1734 cm -1 and 1235 cm -1 correspond to the C----O and P-'~---Ostretching modes
1235 cm 1
1734 cm 1 C=O
0.03
fl
I
~ P=O
! ID
I
~0.02
of the acyl ester linkage and the phosphate group of DMPC, respectively. Incorporation of 20 tool% ot-tocopberol had no significant effect on these stretching bands. This indicates that the carbonyl and phosphate groups do not form hydrogen bonds with the OH-group of a-tocopherol in the phospholipid bilayer. The liposomal zeta-potential, which indicates the membrane potential at the surface, was measured to investigate the interaction of ¢x-tocopherol with the membrane interfacial region. As shown in Table I, the zeta-potentials of EYPC liposomes containing positively charged SA and negatively charged DCP were + 44.3 mV a n d - 42.2 mV, respectively. Incorporation of ascorbyl palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. However, atocopherol had no effect on the zeta-potentials of EYPC liposomes with and without SA or DCP. If the polar groups of ot-tocopherol interact with phospholipid, the motional properties of the 5aposition adjacent to the OH-group of the chromanol should be affected by the membrane surface charge. Table II shows the Ti values of the 13Cand 19F-labeled 5a-position of ot-tocopherol in EYPC- and DPPC-SA and EYPC- and DPPCDCP liposomes. There were no significant differences in the Tl values for 13C o r 19F in these pairs of liposomes with different charges. The OH-group of ot-tocopherol is essential for its intrinsic fluorescence because the fluorescence is lost on acetylation of this group. The location of the OH-group of o~-tocopherol in the phospholipid
I
0
TABLE I 0.01
/ !
!
1800
1750
!
I
1700 1300
\ I
I
I
1250
1200
1150
Effects of ¢-tocopherol and ascorbyl palmitate on zeta potentials of EYPC fiposomes with and without DCP or SA. Zeta potential (mV) EYPC
EYPC-SA
EYPC-DCP
-3.4 -5.0 -22.7
+44.3 +43.9 +!8.9
-42.2 -39.6 -45.4
Wavenumber (cm -1)
Fig. 1. FT-IR spectra of ~ O DMPC
and C ~ O stretching bands of
liposomes with (.... ) and without (
) a-
tocopherol. The concentrations of reagents were 20 mM DMPC, 5 mM a-tocopherol and 10 mM HEPES buffer (pH 7.0). Spectra were measured at 37°C.
None a-Tocopherol Ascorbyl palmitate
Molar ratio; EYPC/a-tocopherol/DCP or EYPC/ascorbyl palmitate/DCP or SA = 10:1:1.
SA = 10:2:1,
72 TABLE II 0.8 Relaxation times (Ti) of the 13C- or 19F-labeled 5a-position of ,,-tocopherol in EYPC or DPPC liposomes charged with DCP or SA.
0.6
2
5a
0.4
HO~C16H33 EYPC a
13C.~-Toc 19F-cx-Toc
°i 0
DPPC b
SA
DCP
SA
DCP
1.053 0.620
0.987 0.657
1.803
1.833
Molar ratio of EYPC or DPPC/t~-Toc/DCP or SA = 40:3:2. aTl(s) measured at 20°C. bTl(s ) measured at 45°C.
bilayer was investigated by the transient-state fluorescence technique with the membrane probes 5-, 7-, 12- and 16-NS. These probes have a nitroxide spin group attached at different positions along the stearic acid hydrocarbon chain, and so become situated at different depths in the hydrophobic interior of the membranes. The quenching effects of the probes on the intrinsic fluorescence of ot-tocopherol in DMPC liposomes are shown in Fig. 2. The linearities of these SternVolmer plots indicate that the quenching process followed a collisional mechanism and that the chromanol nucleus was dynamically distributed in various hydrophobic domains of the membranes at different probabilities. The quenching effects of the probes were in the order 5-NS > 7-NS = 12NS > 16-NS, indicating that the time-averaged location of the chromanol nucleus of ot-tocopherol in a phospholipid bilayer is close to the positions occupied by the spin groups of the probes in this order. Acrylamide, a water-soluble fluorescence quencher known to have a very low capacity to penetrate into phospholipid bilayers [25], did not quench the fluorescence of ot-tocopherol incorporated into DMPC liposomes with or without SA
5
i 10
i 15
i 2O
n-NS (,u,M)
Fig. 2. Stern-Volmer plots of quenching of ~-tocopherol fluorescence by n-NS in DMPC liposomes. O, 5-NS; 0, 7-NS; O, 12-NS; O, 16-NS. The concentrations of reagents were 25/~M ¢x-tocopherol, 250 #M DMPC and 10 mM HEPES buffer (pH 7.0). The fluorescence intensity of ot-tocopherol was measured at 37°C with excitation and emission wavelengths of 296 and 325 nm, respectively. I and I 0 are the fluorescence intensifies with and without quencher.
or DCP (Fig. 3), although it quenched the fluorescence of ot-tocopherol in ethanol solution effectively (data not shown). Bisby and Ahmed [16] reported that the ionic strength of a liposome solution changes the efficiencies of quenching of ot-tocopherol fluorescence by n-NSs by changing the chromanol distribution. However, in our experiments, the quenching efficiencies of 5- and 16-NS were not affected by the membrane surface charge. Moreover the SternVolmer plots of 5-NS and 16-NS in DMPC liposomes were not affected by charging the liposomes with DCP or SA (Fig. 3). McLean and Hagaman [26] reported that changes in the membrane phase and fluidity changed the rate of lipid peroxidation. Therefore, we investigated the oxidation rate of ot-tocopherol in liposomes in different physical states. If the OHgroup of o~-tocopherol is present in the hydrophobic inner region of the membranes, its rate of oxidation should be affected by the phase state of the membrane. Figure 4 shows the oxidation of ottocopherol by the water-soluble radical initiator AAPH in DPPC and DMPC liposomes at 30°C. The oxidation rates in gel state DPPC liposomes
73 5-NS (IIM) 10
5 u
!
0.8 _
15
20
n
[]
u
and liquid crystalline state DMPC liposomes were similar, indicating that the membrane fluidity of hydrocarbon chains in the membrane did not affect the oxidation of ot-tocopherol by radicals that penetrated from the membrane surface.
u A
0.6
Discussion
0.4
Fragata and co-worker [14,15] proposed that the OH-group of c~-tocopherol forms a hydrogen bond with the ester carbonyl group of lipid in the 'hydrogen-belt' - 1 0 A within the membrane, because the effective dielectric constant in the membrane region where the OH-group is located is the same as that of the carbonyl group. On the contrary, Srivastava et al. [9] and Perly et al. [10] concluded from t3C-NMR studies that the chromanol ring of ot-tocopherol is located in the membrane interfacial region near the lipid phosphate moiety. Theoretical calculations by Slivastava et al. [13] predicted that the OH-group of o~-tocopherol forms a hydrogen bond with the phosphate group of phospholipid bilayers. We observed no change of zeta-potentials on incorporation of o~-tocopherol into liposomes with different charges (Table I). This finding indicates that there is no ionic interaction, such as hydrogen bond formation, between the OH-group of ottocopherol and the surface of membranes with a positive or negative charge. We also found that the FT-IR spectra of the C----O and P----O stretching modes of DMPC were not changed by incorporation of,-,-tocopherol into membranes above the phase transition temperature (Fig. 1). These results indicate that the interaction between ot-tocopherol and these polar regions of the phospholipid is not sufficiently strong to shift these stretching bands. Villalain et al. [23] observed spectral changes of FT-IR in the C~-O stretching mode of the phospholipid in the presence of ot-tocopherol and speculated that they were due to perturbations of the C~--C2 bonds of sn-2-acyl chains of phospholipid. The lateral diffusion coefficient of EYPC molecules in a membrane bilayer is reported to be 0.9-1.8 × 10-s cm 2 s -l at 20°C [27], 4.0 × 10-s cm 2 s-l at 31°C [28] and 2.6 × 10-s crn2 s -l at 50°C [29]. On the other hand, this coefficient for
o
0.2
0
(~
I
I
20
I
40 60 Acn/lamide (raM)
I
I
80
100
Fig. 3. Effect of membrane charges on quenching of cttocopherol fluorescence by 5-NS or acrylamide in DMPC liposomes. 5-NS (solid line with open symbols), acrylamide (dotted line with closed symbols). (O,O) DMPC liposomes; (&,b) DMPC-SA liposome, (13,1) DMPC-DCP liposomes. Charged liposomes were prepared as described under Materials and Methods. The concentration of SA or DCP was 50 ~M. Concentration of other reagents and experimental conditions were as for Fig. 2.
20 • o
0
15
8 g
•
0
"U
I
I
I
I
60
120
180
240
Time (min)
Fig. 4. Oxidation of ¢-tocopherol by AAPH in DMPC and DPPC liposomes. O, DMPC liposomes; O, DPPC liposomes. The concentrations of reagents were 50 /~M ct-tocopherol, 1 raM DMPC or DPPC, 10 mM HEPES buffer (pH 7.0) and 20 mM AAPH. Reactions were carried out at 30°C.
74
a-tocopherol in EYPC liposomes was calculated as 4.8 × 10-6 cm 2 s -1 at 25°C [12], which is about 100 times that of EYPC, indicating no rigid interaction between the two, even if they form a hydrogen bond. Recently, Bisby and Ahmed [16] reported that the location of a chromanol group in DPPC vesicles is affected by the ionic strength of the medium. If this is so, the membrane surface charge should affect the location of a-tocopherol and if the chromanol group interacts with the polar membrane matrix, change of the surface charge should change the mobility of the chromanol moiety of a-tocopherol in the membrane. However, the Tl values of the ]3C- and 19Flabeled 5a-position of a-tocopherol obtained from the NMR spectra were similar in phosphatidylcholine liposomes with and without a positive or negative surface charge (Table II). This result supports our above conclusion that there is no strong interaction between the OH-group of a-tocopherol and the polar head group of phospholipid. Bisby and Ahmed [16] reported that the chromanol group of a-tocopherol is located close to the surface of a lipid bilayer in the gel-phase but in the hydrophobic inner region of a bilayer in the flnid-phase. In our experiment, however, there was no difference in the oxidation rates of o~-tocopherol by AAPH in gel- and liquid-phase membranes (Fig. 4). These findings indicate that deep penetration of the chromanol into the membrane bilayer is unlikely. The results of depth-dependent quenching of o~tocopherol fluorescence by n-NSs (Fig. 2) and acrylamide (Fig. 3) indicate that the bulk of the chromanol nucleus is located in a position corresponding to that occupied by the spin group of 5-NS, with less in the region labeled with 7-NS 12-NS and with very little in the membrane core labeled with 16-NS and the polar part of the membrane to allow a water soluble acrylamide to reach it. Aranda et al. [12] also reported a similar distribution of the chromanol of a-tocopherol deduced from fluorescence studies with n-(9anthroyloxy)stearic acids (n-AS) as probes, their efficiencies of energy transfer to the chromanol ring being in the order 7-AS > 2-AS > 9-AS = 12-AS.
If the chromanol ring is usually located in the hydrophobic region relatively close to the membrane surface, as indicated above, lipid peroxyl radicals should also be scavenged in this region where the chromanol head group is located. For this, the lipid peroxyl radical, formed deep in the bilayer membrane, must float toward the surface. Ingold and co-workers [3,30] have suggested that the large dipole moment of the peroxyl radical causes it to move up to the membrane surface. Recently, we confirmed experimentally that lipid peroxyl radicals continuously move up to the surface during their generation in the chain reactions of lipid peroxidation (unpublished data). Takahashi et al. [11] observed that a-tocopherol scavenges the peroxyl radicals close to the spin position of 5-NS most effectively, though they supposed that the OH-group of a-tocopherol is located at or near the interface of the membranes. We conclude that the OH-group of ot-tocopherol forms an antioxidant barrier by lateral diffusion at 100 times the speed of phospholipid molecules in the hydrophobic region close to the membrane surface that is occupied by the spin probe 5-NS and that it effectively scavenges lipid peroxyl radicals floating up from the deep hydrophobic region by donating its hydrogen atom to them. References 1 L.J. Machlin (1980) (Ed.) Vitamin E: A Comprehensive Treatise, Marcel Decker, New York. 2 A.T. Diplock, L.J. Machlin, L. Packer and W.A. Pryor, (1989) (Eds.) Biochemistry and Health Implications: Ann. NY Acad. Sci. 570, 1-555. 3 G.W. Burton and K.U. Ingold (1981) Acc. Chem. Res. 19, 194-201. 4 E. Niki (1989) Vitamin 63, 539-549 (in Japanese). 5 K. Fukuzawa, H. Chida, A. Tokumura and H. Tsukatani (1981) Arch. Biochem. Biophys. 206, 173-180. 6 S. Urano, M. Iida, I. Otani and M. Matsuo (1987) Biochem. Biophys. Res. Comm. 146, 1413-1418. 7 S.R. Wassail, L. Wang, R.C.Y. McCabe, W.D. Ehringer and W. StillweU (1991) Chem. Phys Lipids 60, 29-37. 8 R.J. Cushley and B.J. Forrest (1977) Can. J. Chem. 55, 220-226. 9 S. Srivastava, R.S. Phadke, G. Govil and C.N.R. Rao (1983) Biochim. Biophys. Acta 734, 353-362. 10 B. Perly, I.C.P. Smith, L. Hughes, G.W. Burton and K.U. Ingold (1985) Biochim. Biophys. Acta 819, 131-135.
75 11 M. Takahashi, J. Tsuchiya and E. Niki (1989) J. Am. Chem So¢. 111, 6350-6353. 12 F.J. Aranda, A. Coutinho, M.N. Berberan-Santos, M.J.E. Prieto and J.C. Gomez-Fernandez (1989) Biochim. Biophys. Acta 985, 26-32. 13 S. Srivastava, R.S. Phadke, A. Saran and G. Govil (1986) Int. J. Quant. Chem. 12, 169-181. 14 M. Fragata and F. Bellemare (1980) Chem. Phys. Lipids 27, 93-99. 15 J.G. Lessard and M. Fragata (1986) J. Phys. Chem. 90, 811-817. 16 R.H. Bisby and S. Ahmed (1989) Free Radical Biol. Med. 6, 231-239. 17 V.E. Kagan and P.J. Quinn (1988) Eur. J. Biochem. 171, 661-667. 18 T. Fujii, Y. Hiramoto, J. Tera0 and K. Fukuzawa (1991) Arch. Biochem. Biophys. 284, 120-126. 19 S. Urano, I. Otani and M. Matsuo (1985) Heterocy¢les 23, 2793-2796. 20 I. Kumadald, M. Hirai, M. Koyama, T. Nagai, A. Ando and T. Mild (1989) Synth. Commun. 19, 173-177.
21 22 23 24 25 26 27 28 29 30
K. Fukuzawa and J.M. Gebicld (1983) Arch. Biochem. Biophys. 226, 242-246. R.L. Void, J.S. Waugh, M.P. Klein and D.S. Phelps (1968) J. Chem. Phys. 48, 3381. J. ViUalain, F.J. Aranda and J.C. Gomez-Fernandez (1986) Eur. J. Biochem. 158, 141-147. J.B. Massey, H.S. She and H.J. Pownall 0982) Biochem. Biophys. Res. Comm. 106, 842-847. D.B. Chalpin and A.M. Kleinfeld 0983) Biochim. Biophys. Acta 731,465-474. L.R. McLean and K.A. Hagaman (1992) Free Radical Biol. Med. 12, ll3-119. A.G. Lee, N.J.M. Birdsall and J.C. Metcalfe (1973) Biochemistry 12, 1650-1659. M. Bloom, E.E. Burnell, A.L. MacKay, C.P. Nichol, M.I. Valic and G. Weeks (1978) Biochemistry 17, 5750-5762. P.R. Cullis (1976) FEBS Lett. 70, 223-228. L.R.C. Barclay and K.U. Ingold (1981) J. Am. Chem. Soc. 103, 6478-6485.
69
Location and dynamics of a-tocopherol in model phospholipid membranes with different charges Kenji Fukuzawa a, Wataru Ikebata a, Akira Shibata a, Itsumaro Kumadaki b, Tatsumi Sakanaka c and Shiro Urano c aFaculty of Pharmaceutical Sciences, Tokushima University, 1-78 Shomachi, Tokushima 770, bFaculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-01 and CTokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, ltabashi-ku, Tokyo 173 (Japan)
(Received June 11th, 1992; revision received September 11th, 1992; accepted October 6th, 1992) Studies were made on the position and dynamics of the OH-group of a-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (Tl) relaxation times at the 5a-position of a-tocopherol labeled with 13C-or CIgF3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 tool% ~t-tocopherol, though incorporation of 10 mol% ascorbylpalmitate decreased the zetapotentials of EYPC and EYPC-SA liposomes. The ~ O stretching band (1235 cm-I) of the phosphate group and C-m-Ostretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transforminfrared (FT-IR) spectroscopy, were not changed by incorporation of a-tocopherol. These results suggest that no specific interaction occurred between the OH-group of a-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearicacids (n-NSs) on the intrinsic fluorescence of a-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of a-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface. No difference was found in the oxidation rates of a-tocopherol induced by water-sohible 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) in gel state dipalmitoylphosphatidylcholine (DPPC) liposomes and liquid crystalline state DMPC liposomes, indicating that the OH-group is not located deep in the hydrophobic region. Key words: a-tocopherol; vitamin E; liposomes; spin probe; antioxidant
Introduction ct-Tocopherol (vitamin E) is an important component o f biological membranes and is generally considered to protect the unsaturated fatty acyl Correspondence to: K. Fukuzawa, Faculty of Pharmaceutical Sciences, Tokushima University, 1-78 Shomachi, Tokushima 770, Japan. Abbreviations: AAPH, 2,2 '-azobis(2-amidinopropane)dihydrochloride; DCP, dicetylphosphate; DMPC, diniyristoylphospbatidylcholine; DPPC, dipalmitoylphosphatidylcholine; EYPC, egg yolk phosphatidylcholine; FT-IR, Fourier transforminfrared; HEPES, N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid; NMR, nuclear magnetic resonance; n-NS, n-(Noxyl-4,4-dimethyloxasolidin-2-yl)stearic acid; SA, stearylamine.
chains o f m e m b r a n e lipids from peroxidation [1,2]. It is an amphiphilic molecule consisting o f two functional domains, a c h r o m a n o l nucleus with an O H - g r o u p and a h y d r o p h o b i c phytyl side chain. There is evidence that the polar O H - g r o u p terminates the chain o f peroxidation reactions by trapping free radicals (mainly peroxyl radicals) [3,4], whereas the h y d r o p h o b i c phytyl side chain associates with fatty acid and retains ot-tocopherol in the m e m b r a n e bilayer to stabilize the m e m b r a n e and enhance the effectiveness o f the antioxidant action o f ot-tocopherol [5-7]. There are reports that the O H - g r o u p o f o~tocopherol is located at the m e m b r a n e surface [8-11] or in the m e m b r a n e close to the surface
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
70 [12-15]. Proposals have been made, moreover, that the OH-group hydrogen bonds phosphate [9,10] or carbonyl [14,15] groups of adjacent phospholipids. On the other hand, recent fluorimetric studies demonstrated that the chromanol nucleus of o~-tocopherol is located in the hydrophobic inner region of fatty acyl chains in bilayer membranes in a medium of relatively high ionic strength [16,17] and in monolayer micenar membranes [18]. Here we report studies on the position of the OH-group of ot-tocopherol in phospholipid vesicles using various physical techniques and discuss the dynamics of radical scavenging by txtocopherol in phospholipid membranes. Materials and Methods
Materials o~-Tocopherol (over 99% pure) was supplied by Eisai Pharmaceutical Co., Tokyo. 5-, 7-, 12- and 16(N-oxyl-4,4'-dimethyloxazolidin-2-yl)stearic acids (5-NS, 7-NS, 12-NS and 16-NS) were obtained from Aldrich Chemical Co., Milwaukee, WI. 2,2'-Azobis(2-amidinopropane)dihydrochloride (AAPH) and stearylamine (SA) were from Wako Pure Chemical Industries. N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), dimyristoylphosphatidylcholine (DMPC), dicetylphosphate (DCP) and dipalmitoylphosphatidylcholine (DPPC) were purchased from Sigma Chemical Co., St. Louis, MO. Egg yolk phosphatidylcholine (EYPC) was obtained from Nippon Oil and Fats Co., Ibaraki. All other reagents were of analytical grade. Preparations of a-tocopherol labeled with 13C or 19F at the 5a-position were synthesized as described previously [19,20].
Preparation of liposomes Small unilamellar vesicles (SUV) were prepared as described previously [21] with minor modifications and used for experiments other than the NMR study. Stock solutions of EYPC or DMPC in chloroform with or without SA or DCP were evaporated to dryness under nitrogen in a roundbottom flask. The thin lipid film on the glass wall was dispersed with a Vortex mixer in l0 mM HEPES buffer (pH 7.0) to obtain multilamellar
vesicles (MLV). These MLV were disrupted to SUV by sonication in a Bransonic-12 sonic bath at 40°C. Spin probes were incorporated into the liposomal membranes as reported by Takahashi et al. [11] by adding a solution of SUN containing tx-tocopherol to a thin film of n-NS in a flask and sonicating the mixture at 40°C. Single bilayer liposomes of uniform size and high concentration were required to obtain high resolution NMR spectra. We prepared SUV of about 110 nm diameter as reported previously [6]. A volume of 1 ml of ethanol containing 20 /zraol of EYPC, 1.5/~mol of 13C- or 19F-tx-tocopherol and 1 pmol of DCP or SA was rapidly injected through a Hamilton syringe into 20 ml of Tris-HCl buffer (pH 7.4). The suspension was concentrated to 0.5 mi by Amicon ultrafiltration using an XM-100A membrane with rapid stirring under nitrogen at a pressure of 2 atm/cm 2. The opalescent suspension obtained was applied to a Sepharose 4B column and material was eluted with Tris-HC1 (pH 7.4). Finally 0.5 ml of single bilayer liposome suspension was obtained by combining the liposome fractions and concentrating them in dialysis tubing against Ficol 400 (Pharmacia).
Measurements of 13C- and 19F-spin-lattice relaxation times 13C- and 19F-NMR spectra were recorded at 20°C in a Varian 400 MHz spectrometer using D20 as solvent and sodium 2,2-dimethyl-2silapentane-5-sulfonate as internal standard. Spinlattice (Tl) relaxation times were measured by the inversion recovery technique of Void et al. [22]
Measurement of FT-IR spectra A Perkin Elmer model 1720 FT-IR spectrophotometer was used. The samples were analyzed in a Circle Cell (a cylindrical attenuated total reflectance cell). Experiments were carded out in a frequency range of 800-3200 cra -t, with a resolution of 2 cm -I and data were Fourier transformed and averaged after 30 scans.
Determination of zeta-potential The zeta-potential of liposomes was measured in a LAZER ZEE model 501 apparatus (PEN K E N Co., USA).
71
Oxidation of ~-tocopherol by AAPH DMPC or DPPC liposomes containing ~,-tocopherol were incubated with AAPH at 30°C. The reaction was started by the addition of AAPH. The amount of o~-tocopherol oxidized was measured at room temperature by monitoring decrease of fluorescence intensity (Ex: 297 nm, Era: 326) in hexane after extraction of the ¢x-tocopberol with 5 volumes of EtOH-bexane (1:5, v/v). Results
Results of NMR [6,8-10], electron spin resonance (ESR) [7,11], FT-IR [23] and differential scanning calorimetric [23,24] studies and with fluorescent probes [13,16] have indicated that the chromanol ring of ¢x-tocopherol is located near the polar moiety of the lipid matrix. However, these studies did not show whether polar interaction occurs between the chromanol OH-group of ottocopberol and the phosphate or ester carbonyl group of the phospholipid. Figure 1 shows the FT-IR spectra of DMPC liposomes with and without o~-tocopherol at 37°C. The strong bands at 1734 cm -1 and 1235 cm -1 correspond to the C----O and P-'~---Ostretching modes
1235 cm 1
1734 cm 1 C=O
0.03
fl
I
~ P=O
! ID
I
~0.02
of the acyl ester linkage and the phosphate group of DMPC, respectively. Incorporation of 20 tool% ot-tocopberol had no significant effect on these stretching bands. This indicates that the carbonyl and phosphate groups do not form hydrogen bonds with the OH-group of a-tocopherol in the phospholipid bilayer. The liposomal zeta-potential, which indicates the membrane potential at the surface, was measured to investigate the interaction of ¢x-tocopherol with the membrane interfacial region. As shown in Table I, the zeta-potentials of EYPC liposomes containing positively charged SA and negatively charged DCP were + 44.3 mV a n d - 42.2 mV, respectively. Incorporation of ascorbyl palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. However, atocopherol had no effect on the zeta-potentials of EYPC liposomes with and without SA or DCP. If the polar groups of ot-tocopherol interact with phospholipid, the motional properties of the 5aposition adjacent to the OH-group of the chromanol should be affected by the membrane surface charge. Table II shows the Ti values of the 13Cand 19F-labeled 5a-position of ot-tocopherol in EYPC- and DPPC-SA and EYPC- and DPPCDCP liposomes. There were no significant differences in the Tl values for 13C o r 19F in these pairs of liposomes with different charges. The OH-group of ot-tocopherol is essential for its intrinsic fluorescence because the fluorescence is lost on acetylation of this group. The location of the OH-group of o~-tocopherol in the phospholipid
I
0
TABLE I 0.01
/ !
!
1800
1750
!
I
1700 1300
\ I
I
I
1250
1200
1150
Effects of ¢-tocopherol and ascorbyl palmitate on zeta potentials of EYPC fiposomes with and without DCP or SA. Zeta potential (mV) EYPC
EYPC-SA
EYPC-DCP
-3.4 -5.0 -22.7
+44.3 +43.9 +!8.9
-42.2 -39.6 -45.4
Wavenumber (cm -1)
Fig. 1. FT-IR spectra of ~ O DMPC
and C ~ O stretching bands of
liposomes with (.... ) and without (
) a-
tocopherol. The concentrations of reagents were 20 mM DMPC, 5 mM a-tocopherol and 10 mM HEPES buffer (pH 7.0). Spectra were measured at 37°C.
None a-Tocopherol Ascorbyl palmitate
Molar ratio; EYPC/a-tocopherol/DCP or EYPC/ascorbyl palmitate/DCP or SA = 10:1:1.
SA = 10:2:1,
72 TABLE II 0.8 Relaxation times (Ti) of the 13C- or 19F-labeled 5a-position of ,,-tocopherol in EYPC or DPPC liposomes charged with DCP or SA.
0.6
2
5a
0.4
HO~C16H33 EYPC a
13C.~-Toc 19F-cx-Toc
°i 0
DPPC b
SA
DCP
SA
DCP
1.053 0.620
0.987 0.657
1.803
1.833
Molar ratio of EYPC or DPPC/t~-Toc/DCP or SA = 40:3:2. aTl(s) measured at 20°C. bTl(s ) measured at 45°C.
bilayer was investigated by the transient-state fluorescence technique with the membrane probes 5-, 7-, 12- and 16-NS. These probes have a nitroxide spin group attached at different positions along the stearic acid hydrocarbon chain, and so become situated at different depths in the hydrophobic interior of the membranes. The quenching effects of the probes on the intrinsic fluorescence of ot-tocopherol in DMPC liposomes are shown in Fig. 2. The linearities of these SternVolmer plots indicate that the quenching process followed a collisional mechanism and that the chromanol nucleus was dynamically distributed in various hydrophobic domains of the membranes at different probabilities. The quenching effects of the probes were in the order 5-NS > 7-NS = 12NS > 16-NS, indicating that the time-averaged location of the chromanol nucleus of ot-tocopherol in a phospholipid bilayer is close to the positions occupied by the spin groups of the probes in this order. Acrylamide, a water-soluble fluorescence quencher known to have a very low capacity to penetrate into phospholipid bilayers [25], did not quench the fluorescence of ot-tocopherol incorporated into DMPC liposomes with or without SA
5
i 10
i 15
i 2O
n-NS (,u,M)
Fig. 2. Stern-Volmer plots of quenching of ~-tocopherol fluorescence by n-NS in DMPC liposomes. O, 5-NS; 0, 7-NS; O, 12-NS; O, 16-NS. The concentrations of reagents were 25/~M ¢x-tocopherol, 250 #M DMPC and 10 mM HEPES buffer (pH 7.0). The fluorescence intensity of ot-tocopherol was measured at 37°C with excitation and emission wavelengths of 296 and 325 nm, respectively. I and I 0 are the fluorescence intensifies with and without quencher.
or DCP (Fig. 3), although it quenched the fluorescence of ot-tocopherol in ethanol solution effectively (data not shown). Bisby and Ahmed [16] reported that the ionic strength of a liposome solution changes the efficiencies of quenching of ot-tocopherol fluorescence by n-NSs by changing the chromanol distribution. However, in our experiments, the quenching efficiencies of 5- and 16-NS were not affected by the membrane surface charge. Moreover the SternVolmer plots of 5-NS and 16-NS in DMPC liposomes were not affected by charging the liposomes with DCP or SA (Fig. 3). McLean and Hagaman [26] reported that changes in the membrane phase and fluidity changed the rate of lipid peroxidation. Therefore, we investigated the oxidation rate of ot-tocopherol in liposomes in different physical states. If the OHgroup of o~-tocopherol is present in the hydrophobic inner region of the membranes, its rate of oxidation should be affected by the phase state of the membrane. Figure 4 shows the oxidation of ottocopherol by the water-soluble radical initiator AAPH in DPPC and DMPC liposomes at 30°C. The oxidation rates in gel state DPPC liposomes
73 5-NS (IIM) 10
5 u
!
0.8 _
15
20
n
[]
u
and liquid crystalline state DMPC liposomes were similar, indicating that the membrane fluidity of hydrocarbon chains in the membrane did not affect the oxidation of ot-tocopherol by radicals that penetrated from the membrane surface.
u A
0.6
Discussion
0.4
Fragata and co-worker [14,15] proposed that the OH-group of c~-tocopherol forms a hydrogen bond with the ester carbonyl group of lipid in the 'hydrogen-belt' - 1 0 A within the membrane, because the effective dielectric constant in the membrane region where the OH-group is located is the same as that of the carbonyl group. On the contrary, Srivastava et al. [9] and Perly et al. [10] concluded from t3C-NMR studies that the chromanol ring of ot-tocopherol is located in the membrane interfacial region near the lipid phosphate moiety. Theoretical calculations by Slivastava et al. [13] predicted that the OH-group of o~-tocopherol forms a hydrogen bond with the phosphate group of phospholipid bilayers. We observed no change of zeta-potentials on incorporation of o~-tocopherol into liposomes with different charges (Table I). This finding indicates that there is no ionic interaction, such as hydrogen bond formation, between the OH-group of ottocopherol and the surface of membranes with a positive or negative charge. We also found that the FT-IR spectra of the C----O and P----O stretching modes of DMPC were not changed by incorporation of,-,-tocopherol into membranes above the phase transition temperature (Fig. 1). These results indicate that the interaction between ot-tocopherol and these polar regions of the phospholipid is not sufficiently strong to shift these stretching bands. Villalain et al. [23] observed spectral changes of FT-IR in the C~-O stretching mode of the phospholipid in the presence of ot-tocopherol and speculated that they were due to perturbations of the C~--C2 bonds of sn-2-acyl chains of phospholipid. The lateral diffusion coefficient of EYPC molecules in a membrane bilayer is reported to be 0.9-1.8 × 10-s cm 2 s -l at 20°C [27], 4.0 × 10-s cm 2 s-l at 31°C [28] and 2.6 × 10-s crn2 s -l at 50°C [29]. On the other hand, this coefficient for
o
0.2
0
(~
I
I
20
I
40 60 Acn/lamide (raM)
I
I
80
100
Fig. 3. Effect of membrane charges on quenching of cttocopherol fluorescence by 5-NS or acrylamide in DMPC liposomes. 5-NS (solid line with open symbols), acrylamide (dotted line with closed symbols). (O,O) DMPC liposomes; (&,b) DMPC-SA liposome, (13,1) DMPC-DCP liposomes. Charged liposomes were prepared as described under Materials and Methods. The concentration of SA or DCP was 50 ~M. Concentration of other reagents and experimental conditions were as for Fig. 2.
20 • o
0
15
8 g
•
0
"U
I
I
I
I
60
120
180
240
Time (min)
Fig. 4. Oxidation of ¢-tocopherol by AAPH in DMPC and DPPC liposomes. O, DMPC liposomes; O, DPPC liposomes. The concentrations of reagents were 50 /~M ct-tocopherol, 1 raM DMPC or DPPC, 10 mM HEPES buffer (pH 7.0) and 20 mM AAPH. Reactions were carried out at 30°C.
74
a-tocopherol in EYPC liposomes was calculated as 4.8 × 10-6 cm 2 s -1 at 25°C [12], which is about 100 times that of EYPC, indicating no rigid interaction between the two, even if they form a hydrogen bond. Recently, Bisby and Ahmed [16] reported that the location of a chromanol group in DPPC vesicles is affected by the ionic strength of the medium. If this is so, the membrane surface charge should affect the location of a-tocopherol and if the chromanol group interacts with the polar membrane matrix, change of the surface charge should change the mobility of the chromanol moiety of a-tocopherol in the membrane. However, the Tl values of the ]3C- and 19Flabeled 5a-position of a-tocopherol obtained from the NMR spectra were similar in phosphatidylcholine liposomes with and without a positive or negative surface charge (Table II). This result supports our above conclusion that there is no strong interaction between the OH-group of a-tocopherol and the polar head group of phospholipid. Bisby and Ahmed [16] reported that the chromanol group of a-tocopherol is located close to the surface of a lipid bilayer in the gel-phase but in the hydrophobic inner region of a bilayer in the flnid-phase. In our experiment, however, there was no difference in the oxidation rates of o~-tocopherol by AAPH in gel- and liquid-phase membranes (Fig. 4). These findings indicate that deep penetration of the chromanol into the membrane bilayer is unlikely. The results of depth-dependent quenching of o~tocopherol fluorescence by n-NSs (Fig. 2) and acrylamide (Fig. 3) indicate that the bulk of the chromanol nucleus is located in a position corresponding to that occupied by the spin group of 5-NS, with less in the region labeled with 7-NS 12-NS and with very little in the membrane core labeled with 16-NS and the polar part of the membrane to allow a water soluble acrylamide to reach it. Aranda et al. [12] also reported a similar distribution of the chromanol of a-tocopherol deduced from fluorescence studies with n-(9anthroyloxy)stearic acids (n-AS) as probes, their efficiencies of energy transfer to the chromanol ring being in the order 7-AS > 2-AS > 9-AS = 12-AS.
If the chromanol ring is usually located in the hydrophobic region relatively close to the membrane surface, as indicated above, lipid peroxyl radicals should also be scavenged in this region where the chromanol head group is located. For this, the lipid peroxyl radical, formed deep in the bilayer membrane, must float toward the surface. Ingold and co-workers [3,30] have suggested that the large dipole moment of the peroxyl radical causes it to move up to the membrane surface. Recently, we confirmed experimentally that lipid peroxyl radicals continuously move up to the surface during their generation in the chain reactions of lipid peroxidation (unpublished data). Takahashi et al. [11] observed that a-tocopherol scavenges the peroxyl radicals close to the spin position of 5-NS most effectively, though they supposed that the OH-group of a-tocopherol is located at or near the interface of the membranes. We conclude that the OH-group of ot-tocopherol forms an antioxidant barrier by lateral diffusion at 100 times the speed of phospholipid molecules in the hydrophobic region close to the membrane surface that is occupied by the spin probe 5-NS and that it effectively scavenges lipid peroxyl radicals floating up from the deep hydrophobic region by donating its hydrogen atom to them. References 1 L.J. Machlin (1980) (Ed.) Vitamin E: A Comprehensive Treatise, Marcel Decker, New York. 2 A.T. Diplock, L.J. Machlin, L. Packer and W.A. Pryor, (1989) (Eds.) Biochemistry and Health Implications: Ann. NY Acad. Sci. 570, 1-555. 3 G.W. Burton and K.U. Ingold (1981) Acc. Chem. Res. 19, 194-201. 4 E. Niki (1989) Vitamin 63, 539-549 (in Japanese). 5 K. Fukuzawa, H. Chida, A. Tokumura and H. Tsukatani (1981) Arch. Biochem. Biophys. 206, 173-180. 6 S. Urano, M. Iida, I. Otani and M. Matsuo (1987) Biochem. Biophys. Res. Comm. 146, 1413-1418. 7 S.R. Wassail, L. Wang, R.C.Y. McCabe, W.D. Ehringer and W. StillweU (1991) Chem. Phys Lipids 60, 29-37. 8 R.J. Cushley and B.J. Forrest (1977) Can. J. Chem. 55, 220-226. 9 S. Srivastava, R.S. Phadke, G. Govil and C.N.R. Rao (1983) Biochim. Biophys. Acta 734, 353-362. 10 B. Perly, I.C.P. Smith, L. Hughes, G.W. Burton and K.U. Ingold (1985) Biochim. Biophys. Acta 819, 131-135.
75 11 M. Takahashi, J. Tsuchiya and E. Niki (1989) J. Am. Chem So¢. 111, 6350-6353. 12 F.J. Aranda, A. Coutinho, M.N. Berberan-Santos, M.J.E. Prieto and J.C. Gomez-Fernandez (1989) Biochim. Biophys. Acta 985, 26-32. 13 S. Srivastava, R.S. Phadke, A. Saran and G. Govil (1986) Int. J. Quant. Chem. 12, 169-181. 14 M. Fragata and F. Bellemare (1980) Chem. Phys. Lipids 27, 93-99. 15 J.G. Lessard and M. Fragata (1986) J. Phys. Chem. 90, 811-817. 16 R.H. Bisby and S. Ahmed (1989) Free Radical Biol. Med. 6, 231-239. 17 V.E. Kagan and P.J. Quinn (1988) Eur. J. Biochem. 171, 661-667. 18 T. Fujii, Y. Hiramoto, J. Tera0 and K. Fukuzawa (1991) Arch. Biochem. Biophys. 284, 120-126. 19 S. Urano, I. Otani and M. Matsuo (1985) Heterocy¢les 23, 2793-2796. 20 I. Kumadald, M. Hirai, M. Koyama, T. Nagai, A. Ando and T. Mild (1989) Synth. Commun. 19, 173-177.
21 22 23 24 25 26 27 28 29 30
K. Fukuzawa and J.M. Gebicld (1983) Arch. Biochem. Biophys. 226, 242-246. R.L. Void, J.S. Waugh, M.P. Klein and D.S. Phelps (1968) J. Chem. Phys. 48, 3381. J. ViUalain, F.J. Aranda and J.C. Gomez-Fernandez (1986) Eur. J. Biochem. 158, 141-147. J.B. Massey, H.S. She and H.J. Pownall 0982) Biochem. Biophys. Res. Comm. 106, 842-847. D.B. Chalpin and A.M. Kleinfeld 0983) Biochim. Biophys. Acta 731,465-474. L.R. McLean and K.A. Hagaman (1992) Free Radical Biol. Med. 12, ll3-119. A.G. Lee, N.J.M. Birdsall and J.C. Metcalfe (1973) Biochemistry 12, 1650-1659. M. Bloom, E.E. Burnell, A.L. MacKay, C.P. Nichol, M.I. Valic and G. Weeks (1978) Biochemistry 17, 5750-5762. P.R. Cullis (1976) FEBS Lett. 70, 223-228. L.R.C. Barclay and K.U. Ingold (1981) J. Am. Chem. Soc. 103, 6478-6485.
