Chemistry and Physics of Lipids 14 (1975) 193--199 © North-Holland Publishing C o m p a n y

PMR R E L A X A T I O N T I M E S O F M I C E L L E S O F T H E N O N I O N I C S U R F A C T A N T T R I T O N X-100 AND MIXED M I C E L L E S WITH PHOSPHOLIPIDS Anthony A. RIBEIRO and Edward A. DENNIS* Department of Chemistry, University of California at San Diego, ka Jolla, CalifOrnia 92037 U.S.A. Received August 6, 1974,

Accepted October 7, 1974

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and tile nonionie surfactant Triton X-100 form mixed micellar structures at high molar ratios of Triton to phospholipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A 2 can utilize as substrate. Spin-lattice relaxation times (T 1 ) and spinspin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidylcholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that o f pure Triton X-100 micelles. The T 1 values for the phospholipid in the mixed micelles are f o u n d to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as m e m b r a n e models, b u t the lines are somewhat sharper suggesting the possibility of less anisotropic m o t i o n in the mixed micelles than in the vesicles.

1. Introduction

Mixed micelles of phospholipids and the nonionic surfactant Triton X-100 comprise a useful substrate for the study of the mechanism of action of phospholipase A 2 and other enzymes of lipid metabolism [1] and are formed when Triton is used in the purification of membrane-bound proteins [2]. We have employed 220 MHz pmr techniques [3] and gel chromatography I to characterize Triton micelles and mixed micelles with egg phosphatidylcholine. The cloud point [4] of Triton is lowered in the presence of phospholipid [5], and at temperatures below the thermotropic phase transition [6] of the phospholipids, mixed micelle formation is adversely affected [7]. In order to further characterize the structure of Triton micelles and mixed micelles with various phosphatidylcholines, spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable groups in these compounds have now been determined at a temperature below the cloud point of Triton and above that of the effect of the thermotropic phase transition of the phospholipid. * To w h o m to address correspondence. 1 E.A. Dennis, Arch. Biochem. Biophys. In Press.

194

A.A. Ribeiro, E.A. Dennis, Triton-phospholipid mixed micelles

11. Materials and methods

Triton X-100 (Rohm and Haas) is a polydisperse [8, 9] preparation of p, l-oc~ylphenoxypolyethoxyethanols (1) with an average oxyethylene chain a c CH 3 (1)

b i h ~H 3 H H

CH3--~;-. CH 2 ~' CH 3

g

f

e

X ~ / - O C H 2 CH2(OCH2 CH2)8.50l1

CH 3 H H

length of about 9.5 units [10] ; there may also be some heterogeneity m the hydrophobic moiety (8). Dipalmitoyl phosphatidylcholine (Sigma) (lI) and O [I

x (I1)

y

y

x

~) ~ H2OCCH2CH(CH2)12CH3

CH3(CH2)12CH2CH2COiHC

9

+

CH2OPOCH2CH2N(CH3) 3 O z egg phosphatidylcholine (Schwarz-Mann), which was further purified, were employed in the studies. The pmr samples were deoxygenated by several freeze-pumpthaw cycles. Pmr measurements were made on a JEOL PFT-100 Fourier transform system equipped with a Nicolet 1085 computer and disk. T 1 values were determined from the least squares slopes of the intensities obtained from 180-r-90 pulse sequences employing the PRFT method of Vold et al. [11 ]. Routinely, 28 r values varying between 0.001 and 0.600 sec were obtained for each sample. Line widths {~Ul/2) were measured as the full width at half-height maximum intensity on blownup spectra; field inhomogeneity was taken to be the line width of the HOD peak and this was substracted from the reported results. Peak assignments were based on those previously made at 220 MHz [3].

II1. Results and discussion T 1 relaxation times at 100 MHz for the principal resonance lines of Triton micelles 2 and mixed micelles with egg and dipalmitoyl phosphatidylcholine are given in table 1. Line widths and T 2 relaxation times are given in table 2. It should be noted that the cmc of Triton X-100 is about 0.3 mM [21, 22], so that the monomer concentration of Triton is negligible for the relatively concentrated solutions considered here. 2 I:ootnote see next page.

A.A. Ribeiro E.A. Dennis, Triton-phospholipid mixed micelles

2

t"~c'~F'-t

"~

~

,.= ¢:~

,.0

~

o

0

++

°°°°

~

~ = ~

~

~

~1

8 ooo

195

196

A..4. Ribciro. F..t l)eHnis. ]7"#on phosph(dit;id ml.vcd mtccG'~

Table 2 Line widths and spin-spin relaxation times I'l°~pi Parauleter

Sanlple

a

b

aXe'l/2 (ltz)

Triton Triton + DP PC Triton + Egg PC Sonicated DP PC

1.7 2. l 2.2

4.9

X

y

Z

4.5 ~: I b 4 + 1b

(18) (42°0

17

2.0 2.2 34

Sonicated Egg P(" (18) T~ (see) d

TI/T ~

T1/T_~

4c 2.8

Triton Triton + DP PC Triton + Egg PC

0.19 0.15 0.15

0.065

Triton Triton + I)P PC Triton + Egg PC

1.4 1.8 1.7

1.5

(I.07(I 0.084

0.16 0.15

8.0 7.6

3.0 2.8

Sonicated Egg PC (19) (220 MHz,

20°C)

14

7 (20%) > 20 (80%.)

4

aExpefimentat details same as table 1 ; average reproducibility estimated to be about ± 10%. bDistorted triplet with J = 6 a 1 ltz for both phospholipids; z~uI/2 for center line reported. At 220 MHz with DP PC at 37°C, J = 6 ± 0.5 tlz and AUl/2 = 4.5 -+ 0.5 ttz. CEstimated from graph. dCalculated from AVl/2 = 1/Tr T 2. eCalculated from T 1 and spin-echo T 2 data (19). The T 1 values for all peaks in the T r i t o n X-100 micelles increase w i t h t e m p e r a ture. For peaks a and b at the h y d r o p h o b i c end o f the molecule, T 1 / T ; is small and close to u n i t y suggesting isotropic m o t i o n . Similar ratios o f T 1/T 2 for the h y d r o p h o b i c chain o f a n o t h e r s u r f a c t a n t has b e e n i n t e r p r e t e d to reflect behavior o f a hy2 Podo et al. [12] have reported T 1 values for Triton X-100 at 220 Mftz and they arc similar to those reported here, although they do not agree precisely, perhaps due to the differing experimental conditions including higher magnetic field strengths. It should be noted that their spectral assignments for Triton were similar to those previously reported by our laboratory (3), except for the phenyl protons (peak h and i) which were reversed from our assignment. We assigned the upfield doublet to peak h and the downfield doublet to peak i on the basis of the chemical shifts of toluene (13), t-butylbenzene (13), anisole (14, 15), p-methylanisole (16) and the assignments given in Sadtler [17] for 2-(p-t-pentylphenoxy) ethanol (Sadtler #92 l 3 M), 2-(p-sec-butylphenoxy) ethanol (Sadtler #6663 M), and 2-(p-t-butylphenoxy) ethanol (Sadtler #6664 M). The magnitude of the substituent effects in the model compounds is only consistent with this assignment for tile phenyl protons. Dr. Ndmethy has informed me that he concurs in our assignment and that he has conducted additional experiments which confirm it (F. Podo et al., manuscript in preparation).

A.A. Ribeiro, E.A. Dennis, Triton-phospholipid mixed micelles

197

drocarbon liquid [23]. Kushner and Hubbard [24] have suggested on the basis of viscosity and turbidity studies that Triton micelles contain a large amount of water bound to the polyoxyethylene chain. Studies on related surfactants by Corkill et al. [251 and Clemett [261, who found that the T 1 values of the hydrophobic protons were independent of whether the solvent was H20 or D20, have led to the conclusion that water does not penetrate the hydrophobic core of nonionic micelles to any significant extent. Podo et al. [12] have confirmed that this is also the case for Triton X-100. Thus, work on Triton and related surfactants suggests that the micellar core of Triton X-100 behaves as a hydrocarbon liquid and the polyoxyethylene group is heavily solvated with water. The pmr results reported here support this model. The T 1 values for Triton peaks in both Triton micelles and mixed micelles with both phospholipids are similar. In particular, the nine-proton singlet, which arises from the t-butyl group (peak a) at the hydrophobic end of the Triton molecule, presumably reflects the environment at the center of the micellar core and has a T 1 value which is identical within experimental error in Triton micelles and mixed micelles with both egg and dipalmitoyl phosphatidylcholine. The AUl/~ of this peak is also similar in micelles and mixed micelles and the ratio of T 1/T~-for peak a is consistent with isotropic motion in the hydrocarbon core of the mixed micelles. Thus, it appears that the presence of phospholipid does not greatly alter the structural characteristics of the hydrocarbon core of the Triton micelles. The phospholipid in mixed micelles gives rise to high-resolution spectra with full or nearly-full intensities and narrow line widths in contrast to the spectra obtained with unsonicated multibilayer preparations [27 29]. Thus, the phosphatidylcholine molecules in mixed micelles are clearly in a different motional state than that which occurs in multibilayers. In mixed micelles, the different phospholipid protons have different T 1 values and they are similar to those reported for sonicated vesicle preparations containing a single bilayer of phospholipid [18, 19]. The T 1 relaxations in vesicles have been attributed to trans-gauche isomerizations similar to those which occur in hydrocarbon liquids [19, 30, 31] and this is presumably the mode o f T 1 relaxation of the phospholipid in mixed micelles. In vesicles, there is also a slower motional component which is reflected by the fact that T 2 is significantly shorter than T 1 [19] and this is supported by the frequency dependence o f T 1 [32]. Although this slower component has been considered in terms of vesicle tumbling [33] and lateral diffusion, Chanet al. [31] have summarized the arguments against these explanations. Metcalfe et al. [34] have emphasized intermolecular effects and Horwitz et al. [30] have provided a rationale for the T 1 and T 2 relaxations suggesting "configurationally mobile, yet relatively ordered fatty acids" for the motion of the phospholipid in the vesicles. The pmr spectrum of phospholipid in the mixed micelles [3, 7] appears sharper than published spectra in vesicles. In particular, the terminal methyl group (peak x) gives rise to a resolvable triplet in the mixed micelles as shown in fig. 1, whereas fine structure is not observed in vesicles [18, 19]. T1/T ~ for this peak in mixed micelles is significantly greater than unity, yet less than

198

A.A. Ribeiro. I(_t. Dennis. ]'riton-ptzosl)holipid tni.red micHlcs

,'00 h: I

y/b

(1

Fig. 1. Portion ofpmr spectrum recorded at 220 Mttz and 37o(2for mixed micelles of 40 mM Triton X-100 and 10 mM dipalmitoyl phosphatidylcholine.

T1/T 2 calculated from the data of Horwitz et al. [19]. However, their data was obtained under very different experimental conditions and T 2 values based on line widths are really lower limits on T 2 and are only reliable when the T1/T ~ ratio is close to unity. Thus, while the spectra and line width measurements suggest qualitatively that in mixed micelles, the motion of the phospholipid may be less anisotropic than in vesicles, the precise determination of the state of motion of the phospholipid in the mixed micelles must await direct T 2 and 13C-nmr measurements.

Acknowledgement We wish to thank Drs. John Wright, Robert L. Void, Regitze R. Vold and Karol J. Mysels for pertinent discussions. This work was supported by NSF grant GB19056 and the pmr equipment was purchased with NSF grant GP-32829. A.A.R. is a PHS Predoctoral Trainee of the National Institute of General Medical Sciences (GM-1045).

References [1] [2] [31 [4] [5] [6] [7] [8]

E.A. Dennis, Arch. Biochem. Biophys. 158 (1973) 485 A. Helenius and H. S6derlund, Biochim. Biophys. Acta 307 (1973) 287 E.A. Dennis and J.M. Owens, J. Supramol. Struct. 1 (1973) 165 W.N. Maclay, J. Coll. Sci. 11 (1956) 272 A.A. Ribeiro and E.A. Dennis, Chem. Phys. Lipids 12 (1974) 31 D. Chapman, R.M. Williamsand B.D. Ladbrooke, Chem. Phys. Lipids 1 (1967) 445 A.A. Ribeiro and E.A. Dennis, Biochim. Biophys. Acta 332 (1974) 26 C.R. Enyeart, in: Nonionic surfactants, Surfactant science series, Vol. 1, ed. by M.J. Schick. Marcel Dekker, New York (1967) 44-85 [9] P. Becher, in: Nonionic surfactants, Surfactant science series, Vol. 1, ed. by M.J. Schick. Marcel Dekker, New York (1967) 478--515

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[ 10] Rohm and liaas surfactants, ttandbook of physical properties, CS-I 6 G/cd, Rohm and Haas Co., Philadelphia, 6 [11] R.L. Vold, J.S. Waugh, M.P. Klein and D.E. Phelps, J. Chem. Phys. 48 (1968) 3831 [12] F. Podo, A. Ray and G. N6methy, J. Amer. Chem. Soc. 95 (1973) 6164 [13] F.A. Bovey, F.P. Hood, E. Pier and H.E. Weaver, J. Amer, Chem. Soc. 87 (1965) 2060 [14] H. Spiesecke and W.G. Schneider, J. Chem. Phys. 35 (1961) 731 [15] F.L. Langenbucher, E.D. Schmid and R. Mecke, J. Chem. Phys. 39 (1963) 1901 [16] J. Martin and B.P. Dailey, J. Chem. Phys. 37 (1962) 2594 [ 17] Standard nuclear magnetic resonance spectra, Sadtler Research Laboratories, Philadelphia [18] A.G. Lee, N.J.M. Birdsall, Y.K. Levine and 1.C. Metcalfe, Biochiln. Biophys. Acta 255 (1972) 43 [19] A.F. Horwitz, W.J. Horsley and M.P. Klein, Proc. Natt. Acad. Sci. US 69 (1972) 590 [20] J.H. Noggle and R.E. Schirmer, The nuclear overhauser effect, Academic Press, New York (1971) 233 [21] E.H. Crook, D.B. Fordyce and G.F. Trebbi, J. Phys. Chem. 67 (1963) 1987 [22] A. Ray and G. N6methy, J. Amer. Chem. Soc. 93 (1971) 6787 [23] J.R. Hanson and K.D. Lawson, Nature 225 (1970) 542 [24] L.M. Kushner and W.D. Hubbard, J. Phys. Chem. 58 (1954) 1163 [25] J.M. Corkill, J.F. Goodman and J. Wyer, Trans. Faraday Soc. 65 (1969) 9 [26] C.J. Clemett, J. Chem. Soc. (A) (1970) 2251 [27] S.A. Penkett, A.G. Flook and D. Chapman, Chem. Phys. Lipids 2 (1968) 273 [28] C.H.A. Seiter and S.I. Chan, J. Amer. Chem. Soc. 95 (1973) 7541 [29] G.W. Feigenson and S.I. Chart, J. Amer. Chem. Soc. 96 (1974) 1312 [30] A.F. Horwitz, M.P. Klein, D.M. Michaelson and S.J. Kohler, Annals New York Acad. Sci. 222 (1973) 468 [31] S.I. Chan. M.P. Sheetz, C.H.A. Seiter, G.W. Feigenson, M. ltsu, A. Lau and A. Yau, Annals New York Acad. Sci. 222 (1973) 499 [32] A.C. Mclaughlin, F. Podo and J.K. Blasie, Biochim. Biophys. Acta 330 (1973) 109 [33] E.G. Finer, A.G. Flook and H. Hauser, Biochim. Biophys. Acta 260 (1972) 59 [34] J.C. Metcalfe, N.J.M. Birdsall and A.G. Lee, Annals New York Acad. Sci. 222 (1973) 460

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar stru...
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