Infrared and Raman spectra of phosphatidyl-ethanolamine and related compounds

Chemisto' and Physics of Lipids 14 ( 1975) 113 122 © North-Holland Publishing Company

INFRARED AND RAMAN SPECTRA OF PHOSPHATIDYLETHANOLAMINE AND RELATED COMPOUNDS Hideo AKUTSU and Yoshimasa KYOGOKU Institute for Protein Research, Osaka University, Suita, Osaka 565, Japan

Received July 2, 1974,

accepted October 10, 1974

Infrared and Raman spectra of phosphatidylethanolamine from Escherichia coli, L-~-glycerophosphorylethanolamine and o-phosphorylethanolamine were obtained. Most of the bands were assigned to each vibrational mode based on the N deuteration effect, comparison of the intensity in the infrared and Raman spectra and the depolarization degree measurement in the Raman spectra. The spectra of phosphatidylethanolamine can be interpreted by assuming that the molecule takes the dipolar ionic structure both in non-polar solvent and in solid.

1. Introduction Phosphatidylethanolamine is a main component of phospholipids in the cell membrane ofEscherichia coli. In order ot know the molecular structure and physical properties of the phospholipid with the use of vibrational spectroscopy, it is essential to assign main bands to certain vibrational modes. Although many works on the infrared spectra of lipids and phosphates have been done, only a few are the precise studies on the vibrational spectra of phospholipids [1 6]. In this paper we will present fairly detailed assignments of the vibrational spectra of o-phosphorylethanolanfine (OPEL L-c~-glycerophosphorylethanolamine(GPE) and L-c~-phosohatidylethanolamine (PE). From the analysis of vibrational spectra it has become clear that those compounds are in the dipolar ionic structure in solutions and in solids. The structure of the molecules will be discussed.

1I. Experimental A. Materials Phosphatidylethanolamine was extracted from Eseherichia coli strain B by the method developed by Kanemasa et al. [7]. First, the phospholipid fraction was extracted from wet E. coli with the 2 : I mixture of chloroform and methanol. It was

114

H. A kutsu, Y. K),ogoku. l, ibra[ional spectra of phosphati(]vlethanolamine

separated from the other phospholipids by passing through a silicic acid column. "lhe fraction which corresponds to PE was rechromatographed through the DEAE-cellulose column activated by acetic acid. Its purity was confirmed by thin-layer chromatography. L-c~-glycerophosphorylethanolamine (GPE) was prepared by the degradation of PE according to the method by Ballou et al. [8]. o-Phosphorylethanolamine (OPE) was purchased from Tokyo Kasei Co., and was used without further purification. B. Procedures

Infrared spectra were measured with a Perkin Elmer 621 spectrophotometer. The spectra of OPE and GPE were taken for their D20 and H20 solutions, which were placed between two KRS-5 plates with capillary thickness. The pH of the solutions were adjusted by DCI and NaOD or HCI and NaOH without the addition of buffer solution. The spectra of the KBr pellets of the lyophilized powder from the pH adjusted solution were also observed. Since PE does not dissolve in water, its spectra were taken for tetrachloroethylene and carbon disulfide solutions which were put in KBr fixed cells with 0.1 mm path length. Raman spectra were recorded on a JEOL U-1 Raman spectrometer with the exitation of an argon ion laser line at 5145 A. The aqueous solution of OPE was put in a vertical cell through which the exitation light passes from down to top. The concentration of the solution was about 15% and the total volume of the sample was 0.2 ml. The spectrum of PE was measured by the use of a Spex 1401 spectrometer (by the courtesy of Professor G.J. Thomas, Jr., Southeastern Massachusetts University). Five milligrammes of PE was dissolved into 20/J1 tetrachloroethylene and sealed in a Kimax melting point tube of 1.0 mm inner diameter. The tube was placed perpendicular to the incident beam, 4880 )k or 5145 A argon ion laser line. The depolarization degree was measured by the two exposure method with the use of a half wavelength plate. PE had pale yellow color and weak fluorescence was detected. The solution of GPE gave so strong fluorescence that its Raman spectrum could not be taken. The solution of OPE was titrated with I N HC1 and 0.1 N NaOH by the use of a titrator type TTT 1 (Radiometer Copenhagen). The dielectric constants of the dilute carbontetrachloride solutions of PE (1 5%) were nreasured according to the usual heterodyne beat method by the use of the instrument constructed by Professor T. Shimozawa and coworkers, Saitama University, which has 1 KHz oscillator and 5 ml sample cavity.

Ill. Results and assignments A. o-Phosphorylethanolamine ( OPE)

The salt free form of OPE gave the solution of pH 3 5 depending on concentra-

H. Aku tsu, Y. Kyogoku, Vibrational spectra of phosphatidylethanolamine

I CM- I

3000

l~ F

SOLID

<

R

RED SOLUTION H20 D20

RAMAN SOLUTION

ASSIGNMENT

H20

D20

CM -I

CH2 anti. str, CH2 sy]n, str.

2800

+ NHB deg. def. NH~ sym. def.

1500

115

;500

CH2 sci.

OH bend. PO] anti. str. PO~ sym. str. {c o sir 1 C-C-N+ anti.stl C-C-N+(D) anti O-P-O anti.str. C-C-N+ sym.str.

I

IO00 ~ 4

I000

O-P-O sym. str.

500

500

>

Fig. 1. Vibrational spectra of o-phosphorylethanolamine in salt free form. The dotted line shows the spectrum at higher concentration, anti. str.: antisymmetric stretching mode; sym. str.: symmetric stretching mode; deg. def.: degenerated deformation mode; sym. def.: symmetric deformation mode; sci.: scissoring mode; bend.: bending mode; str. stretching mode. tion, and disodium salt gave about pH 10 solution. The pH of the solutions was adjusted by HC1 and NaOH, and the spectra were recorded for the solutions at pH 4.0, 7.2, 8.3 and 9.6. Their features change following the extent of ionization. Figs. 1 and 2 show the spectra of the salt free form and disodium salt. Two strong bands are observed at 1185 and 1090 cm I in the infrared spectrum of the salt free form, while the Raman line at 1185 cm -1 is weak and the 1078 cm 1 line is very strong. The feature is very resemble to that of the H2PO 4 ion [9] and to those of dialkyl phosphates [ 10, 11 ], both of which have the PO 2 group. Therefore, the phosphate group of the salt free OPE must be in the OI: H - O P - O - R form and the two bands are assignable to the antisymmetric and I: O

I 16

H. ,,!kutsu,

Y. Kyogoku, I "ibrational Sl)tctra o/I)hoslJhatidvletham)laHutm

I

;;

I

!

:,

I-:

l

'

• ~'l "l I r",l U.I -I

k ~ ,t"

~50G' • " / ,. 'u

.

t i

3000 J ;.~,r.

'll

:t

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d

j

J

00 i

--

i

]I~UU 2---

5 Ill 0 -

i ('~k

-i

.1:/~[

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il)ljlj

.

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2 I

Fig. 2. Vibrational spectra of o-phosphorylethanolamine in disodium form. anti. str.: antisymmetric stretching mode; sym. str.: symmetric strelching mode; sci.: scissoring lnode; deg, str.: degenerated stretching mode; str.: stretching mode; rock.: rocking mode. symmetric stretching vibrations of the PO 2 group respectively. Fairly strong bands at 935 and 760 cm -1 in the infrared spectrum of the KBr disk must be related to the stretching modes of the (H) O - P - O (R) group, since the both bands disappear in the spectra of the neutral salt and slightly shift on deuteration. They seem to have character of anti- and symmetric stretching modes, because the former band is strong in infrared and the later is strong in Raman spectra. Similar bands are usually observed for dialkyl phosphates, though their band positions are slightly different because of the difference in bond characters [ 10, 11 ]. The strong band in the 3100 to 2700 cm -1 region consists of a broad band with the maximum at about 2900 cm - I and sharp bands at 2900 and 3000 cm 1. The broad band disappear on the N deuteration and thus it may be due to the NH~

H. Akutsu, K Kyogoku, Vibrational spectra of phosphatidylethanolamine

117

group. The two sharp bands undoubtly arise from the CH 2 stretching vibrations. There are two bands at 1630 and 1540 cm 1, which also disappear on the N deuteration. They are assignable to the NH~ degenerated and symmetrical deformation vibrations respectively [12]. A fairly strong band at 1025 cm 1 must be related to the stretching mode of the C N + bond, because similar bands are generally observed for amino acids in the region from 1150 to 950 cm 1 [1 3]. It might alternatively be possible to assign this band to the C - O stretching mode which also generally appears around here. The former assignment is more probable from the following reasons. The 1025 cm 1 band shifts to 983 cm -1 on the N deuteration and its intensity is sensitive to the pH change of the solution. The phenomena indicate that the 1025 cm 1 band is weakly coupled with the motion of the NH~ group and therefore, the band should originates in the C N + bond. The 1025 c m 1 band is strong in the infrared but weak in the Raman spectrum. Tile line at 883 cm -1 , which also shifts to lower frequency on the N deuteration (857 or 807 cm l), is weak in the infrared and medium in the Raman spectrum. Thus the band at 1025 cm 1 and the line at 883 c m 1 are better to be assigned to the antisymmetric and symmetric modes of the C C - N + group rather than the pure C N + and C C stretching modes. The spectra of the sodium salt of OPE show a typical feature of the dibasic anion of monosubstituted alkyl phosphate [ 10, 11 ] i.e. a strong broad band is seen at 1079 cm -1 and a sharp absorption at 977 cm -1 in the infrared, while the line at 974 cm -1 is the strongest in the Raman spectrum. They are thought to arise from the asymmetric and symmetric stretching vibrations of the PO~ group respectively. The two bands at 1630 cm -1 and 1540 cm -1 which are observed for the salt free form cannot be seen and a new band appears at 1644 cm 1 in the spectrum of the sodium salt. it means that the NH~ group of the salt free form is deprotonated and changed to the NIt 2 group. This band is due to the NIt 2 bending mode. At the same time fairly strong C C--N + absorption at 1025 cm I of the salt free form becomes very weak in the spectrum of the sodium salt. In general the stretching mode of a charged group is strong in intensity compared with that of the uncharged group.

B. L-~-Gl)'cerophospho~lethanolamine ( GPE ) The infrared spectrum of GPE at pH 6.0 is shown in fig. 3. A strong broad band with the maximum at 3500 cm 1 may be due to the OH stretching vibration of glycerol and adsorbed water. Besides it we can see two strong bands at 1225 and 1075 cm 1 which are generally observed for tile neutral salts of dialkyl phosphates [10,11]. As there is no band around 1 3 0 0 c m -1 of t h e P O bond [14], the phosphate group of GPE must be in the ionized form instead of the unionized one. The antisymmetric stretching mode of the PO 2 group of GPE which contains a diester bond gives absorption in a little higher frequency region than that of OPE in acidic form. The amino group of GPE may be also in the protonated form because the band at 980 cm -1 was observed on the N deuteration, which has been assigned to

11 F,

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Fig. 3. Vibrational spectra of L-e~-glycerophosphrylethanolamine. anti. str. antisymmetric stretching mode; sym. str.: symmetric stretching mode; str.: stretching mode. the C C N+(D) stretching in the OPE spectra. The assignment is also supported by tire fact that the degenerated deformation vibration of the NH~ group is seen around 1600 cm 1 partly overlapped with the glycerol and water band, and the symmetrical deformation vibration is seen at 1525 cm I. C L-~-Phosphatidylethanolamine (PE)

The infrared and the Raman spectra of PE are shown in fig. 4 and their assignments are given in table 1. The major difference of the infrared spectrum of the c o m p o u n d from those of OPE and GPE are the appearance of the strong sharp bands at 2920, 1735, 1460 and 1170 cm 1. They are ascribed to the aliphatic ester side chain and are assigned to the CH stretching, C=O stretching, CH 2 scissoring and

H. Akutsu, Y. Kyogoku, l/Tbrationalspectra of phosphatidylethanolamine

l 19

Table 1 Assignments of vibrational spectra of phosphatidylethanolamine. Infrared

Raman

Assignments

Solid -I cm

Solution -1 cm

Solution -1 cm

2950 (s)

2950 (s)

2920 2850 1728 1623 1568 1463

2920 2850 1735 1625 1530 1460

CH 3 deg. str. 2940 (vs) (p) 2900 (s) CH 3 sym. str., NIt~ sym. deg. str. CH 2 antisym, str. 2855 (vs) (p) CH 2 sym. str. (solvent) C - O str. (solvent) Ntt~ deg. def. (solvent) NH~ sym. def. 1470 (sh) (dp) tCH 2 sci. 1440 (s) (dp) ) CIt 2 sci. adjacent to C - O Ctt 3 sym. def. 1310 (m) (dp) 1263 (w) (dp)

(vs) (s) (s) (w) (w) (m)

(vs) (s) is) (w) (w) (m)

1412 (w) 1374 (w)

1412 (w) 1370 (w)

1243 (sh)~ 1215 (s) j

1228 (s)

1172 (m) t 1140 (sh))

1104 (sh)] 1090

1080 (s) 1053 (m) 1013(s) 975 (w) 910 (m)

)~

1230 (w) (dp)

PO2 antisym, str.

1125(w)

CH 2 wag., C O--C antisym, str. and others C Cstr.

1090 (sh)

1097 (m) (p)

PO2 sym. str.

1068 (s)

1075 (m) 1070 (sh)

1160 (m)

1030(m) 980 (sh) 890 (w) 875 (w)

805 (m) 752 (w)

815 (w) 750 (w)

755 (w)

/ C - O str., C O C sym. str. and / others "C C - N +antisym.str. CH 2 rocking C-C N+ sym, str. O - P - O antisym, str. O P Osym. str.

vs: very strong, s: strong, m: medium, w: weak, sh: shoulder p: polarized, dp: depolarized. C - O - C antisymmetric stretching vibration respectively. The weak band at 1412 cm - 1 may be assignable to the scissoring m o d e o f the CH 2 group adjacent to the carbonyl group [15]. A r o u n d 3 0 0 0 cm -1 three peaks are seen at 2950, 2920 and 2850 cm 1 in the infrared s p e c t r u m and at 2940, 2900 and 2855 cm -1 in the R a m a n spectrum. The R a m a n lines at 2940 and 2855 cm -1 are strongly polarized. The peak at 2 9 5 0 c m -1 in infrared s p e c t r u m is assignable to the CH 3 degenerated stretching, the 2920 cm 1 band in infrared to the CH 2 antisymmetric, the 2900 cm -1 Raman line to the CH 3 s y m m e t r i c and 2855 cm I R a m a n line to the CH 2 symmetric stretching vibration respectively. However, it should be n o t i c e d that the NH~ stretching band overlaps

12o

t1+ Akutxu+ }. A3,o+oku. Vtbrational spectra o.! p/tosphatidylethanola,+inc 50@0

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~

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l ~ l ~ ,

I

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r

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i

%

. .'"'~

i

-

..+.

.

I..

.

-j

.....F

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Fig. 4. Vibrational spectra of phosphatidylethanolamine fi'om E. coli. A is the infrared spectrum of KBr disk. B is the infrared spectrum of solution. Tetrachloroethvlene was used as a solvent for the measurement in higher wave number region than 930 cm I and carbon disulfide was used in lower region. The dotted line is the spectrum of N- and O-deuterated sample. C is the Raman spectrum of tetrachloroethylene solution. with these peaks. Strong polarization of tile 2940 cm 1 line may be partly due to the polarization of the overlapped NH~ symmetric stretching mode. The appearence of the band at 1225 cm- l due to the antisymmetric stretching mode of tile PO 2 group indicates that tile molecule is in the ionic structure. The symmetric stretching band of the PO 2 group, which generally appears around 1085 cm I must be overlapped with the C C band due to the skeltal optical mode of the hydrocarbon chains with a motion such that alternate carbon atoms move in opposite directions along the chain axis [5, 6]. If the chains are all trans, the Raman spectrum shows two intense bands at 1130 and 1065 cm 1. The appearence of the band at 1090 cm 1 means that the structure contains several gauche rotations in the paraffin chains. In addition to the broad band at 2940 cm 1, two bands at 1650 and 1580 cm ~ 1 which disappear on the N deuteration show the existence of the NH~ group. The appearance of the band at 1025 cm -1 due to the stretching mode of the C C N + group also supports it. The band at 805 or 815 cm - I in the infrared and the line at 750 cm -1 in the Raman spectra may be ascribed to the O P O antisymmetric and symmetric stretching vibration respectively (9). D. T i t r a t i o n o J" O P E

2 ml of OPE solution in 2.7 mM was titrated by 1 N HC1 and 0.l N NaOH solution and two pK a points were detected at 5.8 and 8.0 in the titration curve.

H. A kutsu, K Kyogoku, Vibrationalspectra of phosphatidylethanolamine

121

E. Dipole moment of PE in CC14 Dielectric constant of the CC14 solution of PE was measured for the four solutions in the concentration range from 1 to 5%. Using electronic polarization obtained from the atomic and bond refractive indices, permanent dipole moment of PE was calculated to be 2.86 debye.

IV. Discussion By comparing the pH dependence of the vibrational spectra and the titration, the pK a at 5.8 of OPE corresponds to the second ionization of the phosphate group and the pK a at 8.0 should be ascribed to the protonation at the amino group. The dipolar ionic structure of PE is generally accepted and Chapman and Morrison confirmed it by NMR study [3]. However Abramson et al. claimed that the infrared spectrum of phosphatidylethanolamine shows the existence of the P - O H group and as a consequence, tile NH 2 group instead of the NH~ one [1 ]. They came to the conclusion from their assignments that strong broad bands at 2800 and 1040 cm 1 arised from the vibration of the P - O H group. However, it is more reasonable to assign the band around 2800 cm 1 to the NH~ stretching mode as discussed above. They assigned the 1040 cm 1 band to the P - O H group referring the pH change of the spectrum of phophatidic acid, a monoester of phosphoric acid. PE and GPE, however, are diesters of phosphoric acid and it is unlikely to compare the spectrum of monoester with that of diester. The band at 1040 cm 1 may be due to the C - C - N + antisymmetric stretching vibration as mentioned above. The dipolar ionic structure is retained both in polar and non-polar solvents. For such a compound we can expect a fairly large permanent dipole moment. The distance from the N + atom to the middle point between two O 1/2 atoms is about 4 A in the crystal of L-c~-glycerophosphorylcholine [ 15 ]. If the charges of plus and minus one electron unit are assumed to locate on the nitrogen and the middle point between two oxygens respectively, we can expect more than 20 debye for the dipolar group. Observed dipole moment, however, is 2.86 debye which is fairly smaller than the expected one. It is difficult to explain it as a result of cancelling the moment of the dipolar group by the moment of the other parts, because triglyceride part only contributes to the moment about 3 debye. Paranjpe and Davar [17] studied the dielectric properties of aliphatic acids and their esters, and got 2.4I debye for oleic acid and 3.38 debye for triolein. Thus it is better to ascribe the small dipole moment to the dimer or micell formation where the moment of each molecule arranges so as to cancell each other. Similar consideration has been made for lecithin which showed almost zero moment in non-polar solvent [I 8].

122

H. Akutsu, ~. IQ,ogoku, Vibrational spectra of phosphatidylethanolamme

Acknowledgements The authors are greatly indebted to Professor T. Shimozawa, Saitama University. and to Professor G.J. Thomas, Jr., Southeastern Massachusetts University, for the use of their facilities.

References (1] [2] [3] [4] [5] [6] [7] [8] [9] [ 10] I 11 ] [12] [ 13 ] [ 14] [15] [16] [17] [18]

B. Abramson, W.T. Norton and R. Katzman, J. Biol. Chem., 240 (1965) 2389 D. Chapman, P. Byrne and G.G. Shipley, Proc. Roy. Soc. A290 (1966) 115 D. Chapman and A. Morrison, J. Biol. Chem. 241 (1966) 5044 D. Chapman, R.M. Williams and B.D. Ladbrooke, Chem. Phys. Lipids 1 (1967) 445 J.L. Lippert and W.L. Peticolas, Proc. Natl. Acad. Sci. U.S. 68 (1971) 1572 J.L. Lippert and W.L. Peticolas, Biochim. Biophys. Acta 282 (1972) 8 Y. Kanemasa, Y. Akamatsu and S. Nojima, Biochim. Biophys. Acta 144 (1967) 382 C.E. Ballou, E. Vilkas and E. Lederer, J. Biol. Chem. 238 (1963) 69 A.C. Chapman and L.E. Thirlwell, Spectrochim. Acta 20 (1964) 937 T. Shimanouchi, M. Tsuboi and Y. Kyogoku, in: Advan. Chem. Phys., vol. 7. Interscience, New York (1964) 435 Y. Kyogoku, T. Shimanouchi and M. Tsuboi, in: Proceedings of the International Symposium on Molecular Structure and Spectroscopy (Tokyo) (1962) A106-1 A. Leifer and E.R. Lippincott, J. Am. Chem. Soc. 79 (1957) 5098 M. Tsuboi, T. Takenishi and A. Nakamura, Spectrochim. Acta 19 (1963) 271 L.J. Bellamy, The infra-red spectra of complex molecules. Methuen, London (1954) R.G. Sinclain, A.F. McKay and R. Norman Jones, J. Am. Chem. Soc. 74 (1952) 2570 M. Sundaralingam, Nature 217 (1968) 35 G.R. Paranjpe and D.J. Davar, Indian J. Phys. 12 (1938) 283 R. Kuhn, I. Hausser and N. Brydowna, Berichte 68 (1935) 2386