water interface

Chemistry and Physics of Lipids, 27 (1980) 1~8 © Elsevier/North-Holland Scientific Publishers Ltd.

IONIC PROPERTIES OF PHOSPHOLIPIDS AT THE OIL/WATER INTERFACE

MAKOTO HAYASHI, TAKONORI KOBAYASHI, TSUTOMU SEIMIYA*, TOSHIO MURAMATSU** and ICHIRO HARA**

Laboratory of Chemistry, College of Arts and Sciences, Chiba Universuty, Yayoicho, Chiba, *The department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Setagaya-ku, Tokyo and **Laboratory of Chemistry, The Department of General Education, Tokyo Medical and Dental University, Ichikawa, Chiba (Japan) Received September 3rd, 1979

accepted January 16th, 1980

Surface pressures and surface viscosities of dipalmitoylphosphatidylethanolamine and its polar group analogues were measured at the hexane/water interface as a function of bulk pH. The monolayers expanded as the bulk pH was shifted from neutral to alkaline and gave rise to an increase in surface pressure at a constant area, and the surface viscosity was simultaneously reduced at high pH. For the increase in surface pressure, theoretical values were calculated using the Gouy-Chapman equation for the electrical double layers produced by shifting pH, and good agreements were obtained with the measured ones from which a simple mechanism was deduced for the increase in pressure. The ionic dissociation characteristics of amino groups of the lipids were discussed taking pKa values given in the above calculations into account. The reduction of the surface viscosity was thought to be attributable to disintegration of zwitter ionic structure in the condensed monolayers.

Introduction S a t u r a t e d d i a c y l p h o s p h a t i d y l e t h a n o l a m i n e gives a clear p l a t e a u r e g i o n o n its surface p r e s s u r e - a r e a curve of the m o n o l a y e r at the o i l / w a t e r i n t e r f a c e as well as at the a i r / w a t e r i n t e r f a c e u n d e r the a p p r o p r i a t e c o n d i t i o n s [1--5]. T h i s fact indicates that it t r a n s f o r m s f r o m a n e x p a n d e d state to a c o n d e n s e d o n e , c h a n g i n g its m o l e c u l a r c o n f o r m a t i o n as is clearly s h o w n b y a n a b r u p t c h a n g e in surface viscosity [5]. This c o n f o r m a t i o n a l c h a n g e is c h a r a c t e r i s e d by a lattice f o r m a t i o n in the c o n d e n s e d films which is d e m o n s t r a t e d b y the e x t r e m e l y high surface viscosity. T h e surface viscosity in the e x p a n d e d films, o n the c o n t r a r y , is as low as those u s u a l l y f o u n d in simple lipids at the h e x a n e / w a t e r interface. H o w e v e r , w h e n it is b r o u g h t i n t o the c o n d e n s e d state p a s s i n g t h r o u g h the p h a s e t r a n s i t i o n r e g i o n f r o m t h e e x p a n d e d state, it gives rise to a n e n o r m o u s l y high value. F r o m this fact, it was p o s t u l a t e d that a n i n t e r m o l e c u l a r l i n k a g e was f o r m e d in the c o n d e n s e d films d u e to a n i n t e r m o l e c u l a r salt f o r m a t i o n b e t w e e n p h o s p h a t e a n d a m i n o g r o u p s [5]. If

2

M. Hayashi et al., Phospholipid monolayers

this is a possible interpretation for this structure, the ionic lattice might be disintegrated when the zwitter ionic structure is unbalanced by shifting pH, which must reflect the surface viscosity in the condensed films, and the expansion of the films must occur simultaneously as the charges appear. With this in mind, the effects of pH in subphase solution on the surface pressure and the surface viscosity were measured at the hexane/water interface.

Materials and methods

L-Dipalmitoylphosphatidylethanolamine, L-dipalmitoylphosphatidyl-Ndimethylethanolamine, L-dipalmitoylphosphatidyl-N-monomethylethanolamine are commercial products purchased from Fiuka AG (Switzerland), and DL-dipalmitoylphosphatidyl-n-butanolamine was synthesised in pure state in our laboratory. Purity of all lipids used in this study was confirmed by TLC as sufficiently pure and they were used without further purification. Monolayers were spread by delivering chloroform/methanol solution (2:8, v/v) of the lipids using a microsyringe (Hamilton) specially devised onto the hexane/water interface, which was formed in a circular glass frame (14 cm diam.) as described in a previous paper [5]. In aqueous subsolution, sodium salt of ethylenediaminetetraacetic acid (EDTA) was added at approx. 10-4 M to prevent possible effect of polyvalent metal ions, and ammonium chloride was also added at 10-2M to maintain a constancy of salt concentration as well as buffering action while adjusting pH in alkaline side to make it easier. Surface pressure were measured by the hanging plate method combined with an electromicrobalance. For the hanging plate, a platinum plate (2.5 c m x 1.0cm) was coated with carbon black to maintain the hydrophobicity of the faces of the plate. The rotational disk method was used for measurements of surface viscosity. A hollowed disk of 3.5 cm in diameter made of teflon was hung using a torsion wire approx. 10 cm long from a synchronous motor rotating at the rate of I rev./min. The distorted angle appeared between the disk and the axis of the motor while measurement was detected on a recorder chart by a specially devised switching system using the circuit shown in Fig. 1. To vary the subphase pH, concentrated ammonium hydroxide solution was added to the aqueous subphase outside the glass frame and gently stirred. After several minutes the stirring was stopped, and the subphase pH, the surface pressure and surface viscosity were recorded.

M. Hayashi et al., Phospholipid monolayers

3

[ SVIIChFOIIOLISI

M2 O/W interface rder Fig. 1. Circuit for switching system. M1, M2: Mieroswitches for starting and reseting the integrator Pl, which are operated by a cam rotating with the synchronous motor. Pz: Amplifier operated by the optical sensor for ending the operation of p~.

Results

and discussion

F i g u r e 2 s h o w s t h e effect of b u l k p H o n t h e p r e s s u r e - a r e a i s o t h e r m of L - d i p a l m i t o y l p h o s p h a t i d y l - N - d i m e t h y l e t h a n o l a m i n e as a n e x a m p l e . T h e i s o t h e r m is s h i f t e d to l a r g e r a r e a s at h i g h e r p H , b e c a u s e of e x p a n s i o n o f t h e film in t h e w h o l e r a n g e . T o o b s e r v e this effect m o r e s i m p l y , t h e s u r f a c e p r e s s u r e a n d t h e s u r f a c e v i s c o s i t y a r e i n t e r m i t t e n t l y m e a s u r e d at a fixed a r e a b y d e l i v e r i n g 1 / z l of t h e s p r e a d i n g s o l u t i o n to t h e c i r c u l a r t r o u g h as !

40 "7K Z

E

~20 U~

Q. O

O CJ

D m0

o

200

Are(] / ~2. molec. 1 Fig. 2. Surface pressure-area isotherms of L-dipalmitoylphosphatidyl-N-dimethylethanolamine at the hexane/water interface at 20°C. The subphase pH values are adjusted by 10-2M phosphate buffer containing 10-4M EDTA. Subphase pH; (1) 9.8, (2) 6.1.

M. Hayashi et al., Phospholipid monolayers

4

mentioned above with respect to the subphase pH values. The open circles in Figs. 3----6 are the measured points of surface pressure, and the surface viscosities are shown by the dashed lines in Figs. 3 and 4. The solid lines in these figures are the theoretically calculated curves as described below. In the alkaline region, the monolayer of phosphatidylethanolamine must be negatively charged by phosphate, because of deprotonation of amino groups, which leads to a rise in surface pressure at a fixed area. As seen in these figures, the surface pressures are raised stepwisely following the bulk S-

b~ >,

8~

\

o

\ \

/,0

\

o

30

s

/

12,-

E

ffl ffl

0

(3) ~

~0 o

1

I

6

I

I

10

PHb Fig. 3. Surface pressure and surface viscosity of L-dipalmitoylphosphatidylethanolamine as a function of bulk pH at the various areas at the hexane/water interface (200C). Aqueous phase contains 10 -2 M ammonium chloride and 10-4M E D T A . Dashed lines, surface viscosity. Open circles, measured surface pressures. (1), Condensed film (44.0 ~ 2 . molec-l), (2), Transition 0 2 o region (81.0 A • molec-1), (3), Expanded film (154 A 2 • molec-l). Solid lines, calculated curves (see text).

M. Hayashi et al., Phospholipid monolayers

5

"7 8. ~

E~

N

40

,E3 0

-o

4~ to 0 O tO

l-j/

0"~

U

Ill to

~,

oo,

S.20

(J'}

I

I

I

6

I

10 PHb

Fig. 4. L-dipalmitoylphosphatidyl-N-monomethylethanolamine (conditions, see Fig. 3). Open circles, (1), Condensed film (42.0/~ 2. molec-Z), (2), Transition region (73.7 ~ 2 . molec-1), (3), Expanded film (94.7 ~2. molec-l). p H (pHb). If this increase in surface pressure results solely from the charges appeared in the films, it is equivalent to free energy of electrical double layer formed under the monolayer. The free energy of double layer is calculated by integration of G o u y - C h a p m a n equation (eqn. 4) as follows, if is valid for this case [6--8]. F = A H = y+ - y_ =

f

~o

tr d~/,

=(21~Tn)'/2.2k-~--[cosh.sinh-'k2--D-~T~n]1] dO

(

,./,FO-2 ~ 1 / 2

(1)

where, F is the free energy of the electrical double layer; AH is the increase in surface pressure; y+, y are the interfacial tension at neutral and high p H respectively; D is the dielectric constant of aqueous phase vicinal to the monolayer; k is the B o l t z m a n constant; T is the absolute temperature; n is the concentration of univalent ions in aqueous phase; ~ is the unit charge;

6

M. Hayashi et al., Phospholipid monolayers

® ® 40

O °

"7 E

4O

_o_oJ

Z

E 30

_o,

o._

o I

I

6

I

I

10 PHb

I

I

I

6

I

10 PHb

Fig. 5. L-DipalmitoylphosphatidyI-N-dimethylethanolamine (conditions, see Fig. 3). Open circles, (1) Condensed film (44.8~ 2. molec I), (2), Transition region (56.3/~ 2- molec J), (3), Expanded film (87.8 ~2. molec-1). Fig. 6. O,L-Dipalmitoylphosphatidyl-n-butanolamine (conditions, see Fig. 3). Open circles, (1), Condensed film (42.0 ]k2. molec-l), (2), Transition region (60.0/~2. molec-1), (3), Expanded film (69.0 A z. molec 1).

and o'0 is the charge density at the charged plane. On the other hand, the acidic dissociation constant, Ka of amino group is expressed by the following equation, when it can be approximated that the dissociation is equilibrated to the surface pH (neglecting the activity coefficients). Atr0H~+ 1 - Atr-------~= Ka

(2)

In this equation, A is molecular area of the film (cm2. mole-l), and H~+ is the hydrogen ion concentration at the charged plane. Hydrogen ion concentration at the surface is connected with that of bulk phase ( H i ) by the following relation according to Gouy-Chapman equation.

H+~ = H i exp(-E~bo/kT)

(3)

where, ~b0 is the Gouy-potential at the surface. As o'0 and ~0 are related by equation 4, we can plot the relationships between increase in surface pressure (All) and the bulk pH (pHb) for the respective systems, making use of pKa as an adjustable parameter:

tro= (2Dk Tn/rr) 1/2 sinh(~tko/2k T)

(4)

M. Hayashi et al., Phospholipid monolayers

7

It is seen in the figures that the measured points coincide well with the theoretical curves thus calculated, except at high pHb. This indicates that the increase in surface pressure is mainly determined by the electrical repulsive pressure, which is shown by the diminished dissociation of amino groups. However, the deviation of the measured points becomes remarkable at high pH, especially at larger areas. This might be caused by changes in molecular conformation accompanying the raised pH or some kind of penetration of solutes into the monolayers by their stronger concentration at high pH, which is sometimes observable in monolayers [9]. The pKa values estimated as described above are ranged between 7.4 and 10.6 (Table I) depending on the chemical species of the lipids and their monolayer states. When comparing these values with pKa of simple alkylamines such as hexadecylamine (10.6) [11], it is seen that they are all considerably small except dipalmitoyiphosphatidyl-n-butanolamine. This difference is thought to be come from either the zwitter ionic structure of the lipids or the fact that they are in monolayer state or both. To clearify this point, dicaproylphosphatidylethanolamine was titrated in aqueous solution by a standard hydrochloric acid solution, and 9.37 was obtained for pKa at 20°C. This lipid is soluble in water and gives a micellar solution, so that the value cited above is considered indicating the intrinsic nature of the polar group of diacylphosphatidylethanolamine. According to phosphorus NMR study of dilauroylphosphatidylethanolamine dissolved in Triton X-100, 9.75 is obtained as an apparent pKa of the amino group [10]. Consequently the low values listed in Table I are thought to be caused not only by the zwitter ionic structure, but also by the fact that they are in a monolayer state. It is worth noting that pKa values are also affected by the type of monolayer, i.e. the condensed state always gives the largest value in every specimen. In Table I, L-dipalmitoylphosphatidyl-N-dimethylethanolaminegives the lowest values of the lipids studied. This is parallel to the fact that tertiary

TABLE I pKa O F A M I N O G R O U P S A T T H E M O N O L A Y E R S

Expanded film Transition region Condensed film

L-dipalmitoylphosphatidylethanolamine

L-dipalmitoylphosphatidylN-monomethylethanolamine

L-dipalmitoylphosphatidylN-dimethylethanolamine

D,L-dipalmitoyl- D,L-dicaproylphosphatidylphosphatidylbutanolamine ethanolamine

8.20

8.30

7.45

9.85

8.35

8.75

7.30

10.0

9.15

9.10

7.90

10.6

9.37

8

M. Hayashi et al., Phospholipid monolayers

amines generally have lower value (9.8) than those of primary and secondary amines [11]. The high values of dipalmitoylphosphatidyl-n-butanolamine will probably be due to its special form of polar group. In Figs. 2 and 3, it is seen that the surface viscosity of the condensed monolayer is decreased following the bulk pH raising, in spite of that the surface pressure increases, and it becomes remarkable as AFI becomes distinguishable. This gives a suggestion in which the high surface viscosity observed in the condensed films must be dominated by their characteristic structure, i.e., two dimensional lattice formation among the zwitter ionic molecules, which has been deduced in the previous papers [5,12]. On the other hand, we found that diacylphosphatidylcholine and sphingomyelin did not show any increase in pressure and also decrease in viscosity even at high pH (<11), though the results are not dipicted here. The high surface viscosity in the condensed films increases further as the surface pressure increases under the same bulk conditions [5]. Therefore, the above results indicate a disintegration of the monolayer structure by the unbalancing of the zwitter ionic structure.

Acknowledgement This work has been financially supported in part by Grant No. 134038 of the Ministry of Education for Scientific Research of Japan.

References 1 B.Y. Yue, C.M. Jackson, J.A.G. Taylor, J. Mingins and B.A. Pethica, J. Chem. Soc. Faraday I, 72 (1976) 2685. 2 J.A.G. Taylor, J. Mingins and B.A. Pethica, J. Chem, Soc. Faraday I, 72 (1976) 2694. 3 M.C. Phillips and D. Chapman, Biochim. Biophys. Acta, 163 (1978) 301. 4 M. Hayashi, T. Muramatsu and I. Hara, Biochim. Biophys. Acta, 255 (1972) 98. 5 M. Hayahsi, T. Muramatsu, I. Hara and T. Seimiya, Chem. Phys. Lipids, 15 (1975) 209. 6 E.J.W. Verwey, J.Th.G. Overbeek and K. Van Nes, Theory of the stability of lyophobic colloids, Elsevier, Amsterdam, 1948, p. 22. 7 J.T. Davies, Proc. R. Soc. Set. A, 208 (195l) 224. 8 J.T. Davies, Trans. Faraday Soc., 48 (1952) 1052. 9 M. Sacre, W. Hoffmann, M. Turner, J. Tocanne and D. Chapman, Chem. Phys. Lipids, 69 (1979) 69. 10 E. London and G.W. Feigenson, J. Lipid Res., 20 (1979) 408. 11 A. Albert and E.P. Serjeant, The determination of ionization constants, Chapman and Hall, London, 1971, p. 92. 12 T. Seimiya, M. Ashida, Y. Heki, T. Muramatsu, I. Hara and M. Hayashi, J. Colloid Interface Sci., 55 (1976) 388.