Nuclear magnetic resonance study of molecular interactions with phosphatidyl choline

Chem. Phys. Lipids 6 (1971) 215-224 © North-Holland Publishing Company

NUCLEAR MAGNETIC RESONANCE STUDY OF MOLECULAR INTERACTIONS WITH PHOSPHATIDYL CHOLINE* GORDON L. JENDRASIAK Department of Physiology and Biophysics, University of Illinois, Urbana, lllinois 61801, U.S.A. 220 MHz N MR spectra of egg phosphatidyl choline (P.C.) in benzene were obtained. The effect of iodine, tetraphenylboron (TPB) and 2,4-dinitrophenol (DNP) on the polar head-group proton signal and the bound water signal was observed. An upfield shift of the proton head group signal was noted for iodine and TPB. All three substances caused line width narrowing. The bound water signal was affected differently by the three substances. The strength of the interaction with the lipid increased in the order DNP
Introduction It has been shown that iodine1,"), 2,4-dinitrophenol a) and tetraphenylb o r o n 3) increase the electrical conductivity of lipids in the bilayer m e m b r a n e form.** Studies of the interaction of these substances with lipid have been made by a d s o r p t i o n spectroscopy 4) and ESRS). In this work, results will be described from nuclear magnetic resonance (NM R) studies. The system phosphatidyl choline in benzene was chosen because considerable work has been done on this system in terms of the nature of the phosphatidyl choline micelle and the water solubilized by the micelle6,7). When egg phosphatidyl choline is placed in benzene at concentrations of greater than 10 -3 M, so-called "large micelles" are formed at 25°C. These micelles consist of perhaps 70-80 m o n o m e r s arranged in l a m i n a r form when less than 5% water is present. W a t e r can be solubilized in the hydrophilic interior of the micelle up to a m a x i m u m of somewhat more than 30% (weight of water: weight of lipid). At c o n c e n t r a t i o n s of P.C. < 10-3 M, small micelles exist with 3-4 m o n o m e r s per micelle. These micelles are p r o b a b l y n o t of bimolecular leaflet structure. This a r r a n g e m e n t of micelles with polar heads exposed to the water presents an interesting system for the study of surface reactions, n o t entirely ** A report of this work was presented at the Biophysical Society Meeting, Baltimore, Maryland, 1970, Abstr. p. 72a. * Abbreviations used: P.C. -- phosphatidylcholine; TPB -- tetraphenylboron; DNP 2,4dinitrophenol. 215

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GORDONL.JENDRASIAK

unrelated to biological systems. It has been shown8), for example, that the rate of I3 formation is greatly increased upon conversion of phosphatidyl choline from the small micelle to "large micelle" configuration. The 13 apparently interacts with the polar-head, water region although it may also be somewhat interior to the hydrocarbon chain region~). N M R techniques would seem to be of value in studying such an interaction. Materials and methods

A Varian model HR-220 MHz nuclear magnetic resonance instrument was used for studying the proton signals. The r scale is used for reporting chemical shifts with TMS (tetramethylsilane) as an internal standard. Control experiments were run to show that the TMS itself did not affect the results. A variable temperature control was used on the instrument. For low concentrations of lipids ( ~ I0 - 4 M), a computer of average transients (CAT) was used when necessary. The temperature of the samples was 23°C_+ I°C, unless otherwise noted. The lipids used were obtained from General Biochemicals and were chromatographically pure. The N M R instrument itself served to check on this purity. Spectral grade CD13, CC14 and C6D6 were used. The iodine, DNP, and TPB were all of reagent grade and were used without further purification. The water used was deionized and twice-distilled in a quartz distillation apparatus. For large micelles, all the spectra were taken for 0.1M egg P.C. in benzene. This assured that greater than 99% of the P.C. was in the form of large micelles. N M R spectra were obtained for the dry samples and the appropriate amounts of water and/or other substances were then added; N M R spectra were then obtained as soon as practical. Time studies were also made by allowing the P.C. samples to remain at room temperature with the appropriate added substance, for 24 hours or more. The phosphorus NM R signal was obtained with the Varian HA-100 instrument operating at 40.5 MHz. A capillary containing (CH30)3P was used as a reference. Correction was made for magnetic susceptibility effects. The CAT was necessary for these measurements. Results

Table 1 summarizes the results of this work. It should be noted that the N(CH3) 3 signal occurs at about 6.25 p.p.m, for " d r y " egg P.C. (0.1M) in benzene and 6.63 and 6.66 p.p.m, in CC14 and CDC13, respectively. Addition of water to dry egg P.C. micelles in benzene causes a considerable narrowing of the N(CH3)3 proton signal as well as an upfield shift of the signal. We have made these measurements over the entire range of 1% to

N U C L E A R M A G N E T I C R E S O N A N C E S T U D Y OF M O L E C U L A R I N T E R A C T I O N S

217

TABLE 1 Summary of N M R results (-N (CH3)s protons) Solvent

[P.C.] 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.t

M M M M M M M M

CDCI3 CDCI:~ CCI4 CCI4 C6D6 C6D6 C6D6 C6D6

5 X 1 0 -4 M

C6D6

5x 5x 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

C6D,~ C~D~ CoD6 C6D, C,iD,~ C6D6 CDC[3 CDCI3 C6D6 C6D~

10 4 M 10 ,1 M M (5-6 % H20) M (5 6% H20) M M M (2-3% H20) M (2-3% H20) M (4% H20) M (4% H20) M (4%o HzO) M (4% H20)

C C h

Substance added 0.1 M I2 0.1 M I2 1% HzO 0.1 M 12 -

Line width (Hz)

0.05 0.05 0.05 0.17

11 6.6 68 35 117 35 106 42

-

5 x 10 4M 12 0.1 M I2 0.1 M TPB 0.01 M TPB 0.1 M TPB 0.1 M D N P -

CCI4

Ar(p.p.m.)

0.08 0.59 1.4 0.12 1.3 <0.01 -

0.1 M DNP

<0.01

*

37 17 ** 64 40 ** 18 13 18 11

* Noise level prohibits meaningful line width measurement; line width is on order o f t h a t for 10 a M 12 case. ** Because the head group signal is superimposed upon signals from other moieties in the lipid, the line width is not given.

30% water addition. Water also causes a narrowing

a n d lesser s h i f t w i t h

CCI 4 as a s o l v e n t . T h e m i c e l l a r - b o u n d w a t e r p r o t o n s i g n a l a p p e a r s a t 4.6 p . p . m , in b e n z e n e a n d 5.05 p . p . m , in C C I 4. Addition narrowing

of iodine to 0.1M "dry"

egg P.C. in b e n z e n e c a u s e s b o t h a

a n d a n upfield s h i f t o f t h e h e a d g r o u p s i g n a l . I o d i n e is m o r e

effective t h a n w a t e r o n a m o l a r b a s i s in c a u s i n g a n upfield shift. In C D C 1 3 o r CC14, i o d i n e a d d i t i o n c a u s e s o n l y a s l i g h t upfield shift. A d d i t i o n o f i o d i n e t o s a m p l e s in b e n z e n e c o n t a i n i n g f r o m 1% t o 3 0 % w a t e r a l w a y s p r o d u c e d a f u r t h e r u p f i e l d s h i f t as well as i n c r e a s e d n a r r o w i n g

of the head group

signal. The addition of water to a "dry" sample to which iodine had been a d d e d a c t u a l l y c a u s e d a d o w n f i e l d s h i f t o f t h e s i g n a l . T h i s is c o n s i s t e n t w i t h t h e lesser e f f e c t i v e n e s s o f w a t e r in c a u s i n g a n upfield s h i f t o f t h e h e a d - g r o u p proton

signal. F o r l a r g e m i c e l l e s in b e n z e n e , t h e w a t e r p r o t o n

shifted upfield upon

iodine addition at low water concentration

s i g n a l is (<5%).

I n C D C 1 3 o r CCI4, t h e w a t e r s i g n a l is u n a f f e c t e d b y i o d i n e a d d i t i o n . Figs.

218

GORDON

L. JENDRASIAK

I'

T %4S

oH,

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A/ jI

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z-J h

40

510

61

i

O

p

/

i

70

80

90

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40

5LO

6LO

70

80 ppm

Fig. I.

Effect of iodine on the N M R

90"

I00

(r)

spectra of egg P.C. (O.IM) in benzene. (a) Dry

(b) 0.1 M I2 added.

la and lb illustrate the effect of adding iodine to a dry egg P.C. sample in benzene. The plots shown in fig. 2 illustrate the effect of varying the iodine concentration. Although the P.C. concentration is 0.1 M, the micelle concentration is on the order of 10 -3 M. Note the slight splitting of the (CHz),-proton signal in benzene; this splitting was not observable in the other solvents used. Heating the sample from 25 ° to 40°C and then to 60°C had little effect on the splitting even though the micelle became progressively smaller (70-80 monomers at 25°C and 50-60 monomers at 40°C). Water also appeared to have little effect on this splitting. The splitting is increased by the addition of iodine. It is tentatively suggested that the signal further upfield of the pair is due to the/~-chain of the lipid while the downfield signal arises from the ~'-chain.

NUCLEAR

MAGNETIC

RESONANCE

STUDY

OF MOLECULAR

I

219

INTERACTIONS

I

06

05

12 o. o

"E04

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Fig. 2.

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I i0-~ (M)

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6

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Effect of iodine concentration on the head group proton N M R signal position and line width; large P.C. micelles in benzene. i TMS i

t

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2 N'(CH,), I¸¸

'~'L"","~&,':*"*"?"('.'¢ ' 4¢J

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9~0

I00

5 × 10 a M egg P.C. in benzene. "Small micelles".

In the case of small micelles at 25°C in benzene, the head group is shifted upfield some 0.2 p.p.m, from its position in the large micelle case. More striking, however, is the very prominent splitting of the (CH2),-proton signal as shown in fig. 3. This splitting is increased as the sample is heated such that the configuration goes from tetramer to dimer and finally to monomer. Addition o f iodine to any o f these configurations causes a head group signal narrowing, an upfield shift and an increase in the (CH2) . signal

220

G O R D O N L. J E N D R A S I A K

splitting. On the basis of the ratio of iodine concentration to lipid concentration, the upfield shift of the head group signal is somewhat less than for the large micelle case. When TPB is added to the large micelles, a large upfield shift of the N(CHa) 3 proton signal is observed both in C6Do and CDCI 3. This shift is much larger than for an equal amount of iodine. In addition, the water proton signal was shifted upfield ; again this shift is greater than for iodine. Some water in the micelle structure appears to be necessary to solubilize TPB; TPB is not very soluble in C6D6. Fig. 4 illustrates these effects. Addition of D N P to dry P.C. micelles in C6D6 causes neither a narrowing of the head group signal nor a shift; D N P is soluble in CoD 6. When the same amount of D N P is added to micelles containing water in C 6 D 6 or CC14, a narrowing of the N (CH3) 3 proton signal is observed but no shift. In both (CH21

FJ'I(:H.I

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,

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90

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Fig. 4.

Effect of TPB on the N M R spectrum of egg P.C. (0.1 M) in benzene. (a) 5-6 ~ (b) 5 - 6 ~ H~O + 0 . 1 M TPB.

H20

N U C L E A R M A G N E T I C R E S O N A N C E S T U D Y OF M O L E C U L A R I N T E R A C T I O N S

221

C6D 6 and CCl 4, the water proton signal is shifted downfield upon D N P

addition. Dipalmitoyl phosphatidyl choline was also studied at concentrations of 5 X 10 - 4 M at 31°C, 1 x 10 3 M at 33°C and 1 x 10 -z M at 57°C, in C6D 6. In all cases, addition of iodine caused an upfield shift of the head group signal, indicating that the double bond is not necessary for the observed effects. The splitting of the hydrocarbon chain protons was observed at 5 X 10 - 4 M concentration in C6D,, indicating the splitting is not due to the double bond. The phosphorus N M R signal (0.IM P.C.) was found to be somewhat narrower in CC14 than in C6D 0. Iodine narrowed the signal slightly in C6D6 but not in CCI4. In neither solvent was a chemical shift of the signal observed upon iodine addition. Discussion

The upfield shift of the N (CH3)3 proton signal, upon addition of iodine, water or TPB, can be ascribed to several factors: these include interaction with the lipid, displacement of solvent from the polar head group region and an alteration of the micelle structure. Our results indicate that the micelle is not broken up upon addition of any of the substances discussed in this work. For iodine, it has been shown that the CMC value, in various solvents, obtained by measuring I~formation is essentially the same as that obtained using physical techniques (ref. 8). From figs. 1 and 3, the splitting of the hydrocarbon chain proton signal is quite different for the case of large P.C. micelles as opposed to small micelles in C6D6. Addition of iodine to large micelles may increase the splitting but at no time does it resemble that for the tetramer or smaller forms of the lipid; D N P and TPB do not affect the splitting. Moreover, the line-width narrowing and upfield shift occur, at least for the iodine case, when the egg P.C. is present in the tetramer, dimer or monomer form. Water, which acted in a sinular manner to iodine, has been shown to be solubilized by egg P.C. micelles in benzene up to 25% (g water: g lipid) without changing the number of monomers per micelle. We, therefore, conclude that the addition of iodine, DNP or TPB to large egg P.C. micelles in C6Do (and by inference in CDC13 and CC14) does not result in a breaking up of the micelle structure. The narowing and upfield shift of the head group signal thus cannot be due to this factor. It is likely, however, that the micelle structure, and, in turn, the head group orientation, are altered due to the necessity of accommodating molecules such as TPB near or in the micelle interior. The fact that the head group proton signal in C 6 D o is downfield from its

222

G O R D O N L. J E N D R A S I A K

position in CC14 or CDCI 3 may well be due to the presence of C6D 6 molecules near the head group region. It has been suggested from N M R studies 9) that benzene is solubilized by cetyltrimethylammonium bromide (CTB) micelles in water at the micelle-water interface. We suggest a similar solubilization for egg P.C. micelles in C6D 6 with the interface now being at the micelle interior. The downfield signal in C6D 6 would indicate that the N ( C H 3 ) 3 protons are located at the periphery of the benzene ring rather than within the ring. The position of the phosphorus N M R signal indicates that phosphorus may, perhaps, not be exposed to the benzene. It has been reported that for egg P.C. bilayers in an aqueous system~°), the head group is oriented parallel to the plane of the bilayer. For egg P.C. micelles in C6D6, the head group is interior to the micelle and could be bent inward, toward the hydrocarbon region, even more severely than tbr the aqueous case, resulting in exposure of the head group to benzene with the phosphorus protected from the benzene. Iodine causes a much greater upfield shift of the head group signal in C6D 6 than in CDCI 3 or CC14. We ascribe this, at least in part, to displacement of C6D6 molecules from the head group region. Iodine interacts with lipid micelles 8) in C6D6, CCI 4 and CDC13 to produce I~-. This interaction could result in displacement of CoD 6 from the head group causing an upfield shift of the N M R signal. In addition, an interaction between 13 and N + would be expected to result in some upfield shift of the head group signal. Although the same type of interaction occurs in CCI 4 and CDCI 3, the shift is much smaller due to the absence of the benzene "ring currents". Water also interacts with the polar head group and might be expected to result in C6D 6 displacement and an upfield shift of the N M R signal. The narrowing of the N M R signal, upon water addition to the micelles, is consistent with the observation that hydration of dry egg P.C. increases the motional freedom of the polar head groupn). Since D N P does not produce an upfield shift of the head group signal, we interpret the DNP-lipid interaction as being weaker than the iodine-lipid interaction. Since the line width narrowing occurs for P.C. micelles with occluded water but nor for " d r y " micelles, it is suggested that the ionized form of D N P interacts with the lipid. The fact that DNP, H 2 0 and iodine all cause a narrowing of the head group signal in various solvents certainly suggests a rearrangement of the micelle interior. The shift in the water signal due to D N P and iodine addition supports this. If this rearrangement produced the upfield shift of the head group signal, it is curious that the shift should be greater in benzene. This would seem to indicate that benzene displacement may be the primary cause of the shift caused by iodine and water.

NUCLEAR MAGNETIC RESONANCE STUDY OF MOLECULAR INTERACTIONS

223

TPB causes essentially the same upfield shift for P.C. both in C 6 D 6 and CDC13, We conclude that the much larger shift observed with TPB is due to both a stronger interaction between the lipid and TPB and the presence of the bulky TPB molecule in the micelle interior. At present, we cannot differentiate between the two effects. Certainly the large shift in the bound water signal with TPB argues for some rearrangement of the micelle structure. TPB would be expected, however, to complex strongly with the N + moiety. TPB is insoluble in C 6 D 6 and with " d r y " P.C. micelles, will not readily interact. This suggests an interaction between the N + and the T P B - ions. As with I3-, a complexing between a negative ion and the N + would be expected to result in an upfield shift of the N + (CH3)3 proton signal. Any small shift due to benzene displacement would be masked by these effects for TPB. We feel that in view of the well known complexing ability of T P B with quaternary a m m o n i u m ions, the large upfield shift with TPB represents, in part, a stronger interaction than for iodine. Iodine, D N P and TPB have all been found to decrease the electrical resistance of lipid bilayer membranes where, it has been postulated, these substances act as charge carriers across the membrane3). The order of effectiveness in decreasing the resistance was found to be T P B > i o d i n e > DNP. If the upfield shift of the N ( C H 3 ) 3 proton signal is taken as an indication of the interaction of these substances with the lipid, our results are consistent with this order. If these substances do act as charge carriers across the bilayer, an interaction of the substance with the polar head group at the bilayer-water interface might be the first step in the conduction process. For egg P.C. bilayer membranes, an increase of both the electrical conductance and capacitance have been found upon exposure of the membrane to iodine12). Solid state films of egg P.C. show a simultaneous increase in electrical conductivity and capacitance upon exposure to iodine or water vapor2). These capacitance increases could be due to an increase in the dielectric constant of the lipid; this increase would be consistent with a less hindered movement of the head group signal as indicated by the line width narrowing of this work.

Acknowledgments The author thanks the University of Illinois Research Board for a grant supporting this work. The National Science Foundation provided funds for the purchase of the Varian H R-220 magnetic resonance spectrometer. The technical assistance of Mr. Robert Thrift was invaluable during the course of this study.

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References 1~ P. L/iuger, W. Lesslauer, E. Marti and J. Richter, Biochim. Biophys. Acta 135 (1967120 2) G. L. Jendrasiak, Ph.D. Thesis, Michigan State University (1967); also published as report AD657954, Defense Documentation Center, Alexandria, Virginia 3) E. A. Liberman and V. P. Topaly, Biochim. Biophys. Acta 163 (1968) 125 4) B. Bhowmik, G. L. Jendrasiak and B. Rosenberg, Nature 215 (1967) 842 5) G. L. Jendrasiak and R. Hayes, Nature 225 (1970) 278 6) P. H. Elworthy, J. Chem. Soc. (1959) 813 7) P. H. Elworthy and D. S. Mclntosh, J. Phys. Chem. 68(1964) 3448 8) G. L. Jendrasiak, Chem. Phys. Lipids 4 (1970) 85 9) J. C. Eriksson and G. Gillberg, Acta Chem. Scand. 20 (1966) 2019 10) T. Hanai, D. A. Haydon and J. Taylor, J. Theoret. Biol. 9 (1965) 278 I 1) D. M. Small, J. Lipid Res. 8 (1967) 551 12) P. L/iuger, J. Richter and W. Lesslauer, Ber. Bunsenges. Phys. Chem. 71 (1967) 906