Langmuir 1991, 7, 207-208
207
Notes Effect of Alamethicin on Stearate Monolayer Igor Vodyanoyt Department of Physiology and Biophysics, University of California Irvine, Irvine, California 9271 7 Received January 3, 1990. I n Final Form: June 11, 1990
The antibiotic alamethicin is recognized as a membraneactive peptide that induces a highly voltage dependent conductance in lipid bilayers' and in biological memb r a n e ~ .At ~ ~present, ~ it can be synthesized and highly p ~ r i f i e dand , ~ its behavior in bulk solutions (water and organic) and in bilayers is well described (for review, see refs 5 and 6). However, there is very little quantitative information about alamethicin-lipid interactionsat water/ lipid interface^.^ We have studied the interaction between purified alamethicin and lipid by measuring changes in the surface pressure-area curve of a monomolecular stearate film induced by alamethicin in the aqueous subphase. The aim of this study is to determine how alamethicin partitions from water into the monolayer. Experimental Section Stearic acid (octadecanoic acid CHs(CH2)&02H) was purchased from Applied Science Laboratories, Inc., PA (No. 3294). The major fraction of alamethicin (denoted fraction 4 from the number of its chromatography peak) obtained after purification of the natural product (Upjohn Co.) by high-pressure liquid chromatography4 was used in these studies. Water for the subphase solutions was redistilled twice. All other chemicals used were reagent grade. Subphase composition was 3 X M CaClz and 2 X M NaHC03 (pH 7.2). All experiments were carried out a t room temperature (22 "C). The monolayer trough was made from a solid piece of highdensity Teflon (9.8 X 25 cm inside dimensions) and enclosed in the glass chamber. A Teflon barrier for controlling the area of the film was driven by a reversible electric motor. The monolayer was compressed with the constant rate of 1 mm/min. The film balance was calibrated in the range 0-80 dyn/cm to an accuracy of f0.1 dyn/cm. Surface pressure was measured by the Wilhelmy method using a filter paper plate8 of dimensions 1 X 2 cm. A methanol solution of alamethicin (from a stock solution 3.4 mg/mL) was added to the subphase solution and thoroughly stirred. Then the mixture was poured in the trough and equilibrated for 15-20 min. The surface was carefully swept to remove surface impurities, and immediately after that a hexane solution of stearic acid (9.1 X 10-2 mg/mL) was added to + Address for correspondence: Office of Naval Research, 800 N. Quincy St., Arlington, VA 22217-5000. (1) Mueller, P.; Rudin, D. 0. Nature (London) 1968,217,713. (2)Sakmann, B.; Boheim, G. Nature (London) 1979,282,336. (3)Cahalan, M. D.;Hall, J. E. J. Cen. Phys. 1982,79, 411. (4)Balasubramanian, T. M.; Nancy, C. E.; Kendrick, M.; Taylor, G. R.; Marshall; Hall, J. E.; Vodyanoy, I.; Reusser, F. J. Am. Chem. SOC. 1981,103, 5127. ( 5 ) Latorre, R.; Alvares, 0. Physico. Reo. 1981,61,77. (6)Hall, J. E. Channels inBlack LipidFilms. InMembrane Transport in Biology; Giebisch, G., Tosteson, D. C., Ussing, H. H., Eds.; Concepts and Models; Springer-Verlag: Berlin, 1978;Vol. 1, pp 475-531. (7)Chapman, D.;Cherry, R. T.; Finer, E. G.;Hauser, H.; Phillips, M. C.; Shipley, G. G. Nature (London) 1969,224,692. (8)Kuhn, H. In Techniques of Chemistry VI. Physical methods of chemistry; Arnold Weissberger, A., Ed.; Spectroscopy of Monolayer Assembly, 1974.
spread a monolayer. The same amount (90 pL) of stearic acidhexane solution was spread in every experiment.
Results Surface pressure vs stearate monolayer area was measured as a function of alamethicin concentration in the subphase. Alamethicin concentration varied from 8 X 10-8 to 8 X M. The number of stearic acid molecules, N,, spread was 1.74 X 10'6for all pressure-areacurves studied. These results are shown in Figure 1 as a series of pressure-area curves. Increasing the alamethicin concentration causes an increase in the apparent area per stearate molecule at low pressure (curves 2-5). At a pressure of about 25 dyn/cm, the area per molecule of films containing alamethicin in the subphase collapses to nearly the area per molecule of a stearate film without the alamethicin (curve 1). The pressure at which this collapse occurs is essentially unaltered by changes in the alamethicin concentration. We were unable to detect an adsorbed alamethicin monolayer on the water phase-air interface at any concentration of antibiotic in the water phase from 8 X to 8 X M; that is, there was no change in surface tension from that of the subphase before an addition of stearic acid to the interface. Discussion If there is an equilibrium between adsorbed and subphase alamethicin, we can write KI
A+S+SA K2
where A is the alamethicin, S is a free stearate, and SA is the alamethicin "bound" to stearate. We assume that adsorbed alamethicin can be in two states: the first state is favored at low pressure and has an area per molecule Sal; the second state is favored a t high pressure and has an area per molecule Sa2. Then the difference between partial areas (per stearate molecule) of a monolayer with adsorbed alamethicin in states one and two can be expressed as where SIand SZare areas of monolayer in states one and two correspondingly. 8 = n,,/N, is the fraction, of the monolayer occupied by alamethicin ( n , is the number of alamethicin-stearate interactions in (l)),and Sa = Sa1 &2.
If alamethicin adsorption obeys the Langmuir isotherm, one can write (3)
where K = K1/Kz is the adsorption coefficient, assumed to be independent on alamethicin concentration in the subphase and on the surface pressure of the monolayer, and [A] is the alamethicin concentration in the subphase. Combining (2) and (31, we get
Q/[AI = KSa - KQ where Q = (SI- S2)/Ns.
This article not subject to U.S. Copyright. Published 1991 by the American Chemical Society
(4)
Notes
208 Langmuir, Vol. 7, No. 1, 1991
Table I. Parameters Calculated from the Experiments Illustrated in Figure 1 curve 2 3 4 5
[A], g/mL x 107 1.36 2.69 6.8 13.6
Q/[A],A2 mL/g
Q,A2 13.5 24.8 32.1 43.9
X
lo-? 0, %
9.93 9.21 4.72 3.23
24.1 44.2 57.2 78.2
U ar
0
c
3
Q
ul
x
-0 0000
0 200
0400
0 600
0800
1000
1200
0
8o 70
2
Monolayer Area, (fraction)
L
0
50 6o
Figure 1. Surface pressure as a function of stearate monolayer area and alamethicin concentration in the subphase. The monolayer was compressed with the constant rate of 1 mm/min in the trough of 25 X 9.8 cm. A methanol solution of alamethicin was added to the subphase prior to the monolayer formation: (1) no alamethicin was added to the subphase; (2) 1.36 X W ; (3) 2.69 X 10-7; (4) 4.6 X 10-7; (5) 1.36 X 10-6 g/mL of alamethicin in the subphase.
40
4 1
LL
20
i/
lo O l ’ !
0 20
I
I
, 2
,
I
4
I
1
6
1
1
8
,
, 10
I
, , , , 12 14 16
Alamethicin Concentration, (mg/ml)
I
*
I
II
18
4
10
Figure 3. Alamethicin adsorption isotherm. Solid line is eq 2. Squares are calculated 0 = Q/S. from Table I. +
1aJ
in surface pressure at 320 A2/molecule and collapse at about 30 dyn/cm a t 200 A2/molecule. They stated that addition of alamethicin in nanomolar concentrations to the substrate below a lipid monolayer caused a penetration of alamethicin into the monolayer as long as pressure was below 30 dyn/cm (area/lipid molecule 60-110 A2). Our phenomenological treatment of pressure-area curves shows that alamethicin adsorption at low surface pressures can obey a simple Langmuir isotherm We found an area of about 80 A2 for an alamethicin molecule in a stearate monolayer under a pressure lower than 25 dyn/cm. When the pressure is increased above 25 dyn/cm, the alamethicin either desorbs from the monolayer or assumes some conformation having a much lower area than 80 A2. We are also unable to distinguish between the following three alternatives; alamethicin forming an immiscible m ~ n o l a y e r alamethicin ,~ binding to stearate, or alamethicin forming a monolayer miscible at low pressure but immiscible at high pressure. An alteration in alamethicin conformation, probably changing from a form with the long axis of the molecule parallel to the membrane surface to one with the long axis perpendicular to the membrane surface, is thought to occur when alamethicin goes from the nonconducting to the conducting state in lipid bilayers.lOJ1 The change we see in monolayer area at high pressure could be associated with such a conformational change in the alamethicin monomer.
4
u
\
0
01
10
20
30
40
50
Figure 2. Left-hand side of eq 4 (Q/ [A]] = KS, - KQ) is plotted against Q. Values of Q = (SI- Sz)/N8.andQ/[A] are shown in Table I. Linear regression line (regressioncoefficientr2 = 0.978) has a slope which determinesa value of the adsorption coefficient (K = 2.44 X 103 mL/mg) and intersection with the Q/[A] axis at 56.1 A2,which represents the difference between the adsorbed alamethicin molecule area under the low- and high-pressure monolayer. We can plot the left-hand side of (4) against Q. Such a plot (Figure 2) gives a straight line with a slope which yields a value of the adsorption coefficient K = 2.44 X lo3 mL/mg, which corresponds to the value of the standard free energy of adsorption (AG) of 8.9 kcal/M. Intersection of this line with the Q/[A]axis gives us a value for S a of 56.1 f 2 A2. Table I represents calculated values of Q, Q/[A], and 0 = Q/S,for all measurements. Because the pressure at which collapse of the monolayer occurs is essentially unaltered by changes in the alamethicin concentration (corresponding area per molecule is 28 f2 A2) and approximately equal to the collapse pressure of the stearate monolayer (corresponding area per molecule is 25 f 1.8 A2,we can calculate the area of alamethicin in the low-pressure state
Sa,= 56.1 + 25.0 = 81.1 f 4.0 A2 An adsorption isotherm calculated by using (2) is shown in Figure 3. The squares are the 0 values from Table I. Chapman et al.7 reported that alamethicin formed insoluble monolayers a t the air-water interface when spread from hexane/ethanol. They found an initial rise
’
Acknowledgment. This work was supported by a ONR SORP Grant and by NIH Grant 30657. Registry No. Alamethicin, 27061-78-5; stearic acid, 57-11-4. (9) Gainea, G. L.,Jr. In Insoluble monolayers at liquid-gas interfaces; Prigogine, I., Ed.; 1966. (10) Boheim, G . J . Membr. Biol. 1974,19, 277. (11) Vodyanoy, I.; Hall, J. E.; Balaaubramanian, T. M. Biophys. J . 19RI. 71. . - - -, 42. __, . -. (12) Hall, J. E.;Vodyanoy, I.; Balasubramanian, T.M.; Marshall, G. R.Biophys. J . 1984,45, 233.
