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Monitoring the permeability profile of lipid membranes with spin probes
ARCHIVES
OF BIOCHEMISTRY
Monitoring
AND BIOPHYSICS
the Permeability
S. SCHREIER-MUCCILL0,2 Division
of Biologicat
17&l-11(1976)
Profile of Lipid Membranes Probes’ D. MARSH,3
AND
Sciences, National Research Council of Canada, Received March 31, 1975
with Spin
I. C. P. SMITH Ottawa,
Canada KlA
OR6
The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows firstorder kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile, The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the halftime of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.
The permeability characteristics of biological membranes have been the object of much study. A large amount of theoretical work dealing with transport phenomena across membranes has been undertaken (see, e.g., Ref. (l-4)). Experimental studies have been performed both on biological and model membranes (5, 6). Electrical measurements on black lipid membranes have been used to relate such properties to permeability characteristics (7-9). Liposomes have been widely employed as model systems for the study of permeabil-
ity (10-12). In this case radioactive permeants have been used (13) as well as electrophoretic mobility data (14) and other electrical measurements (15). In a number of cases comparison has been made between permeability properties of biological membranes and of tryptosomes formed from lipid extracts of such membranes (16). The work concerned with permeability properties of lipids has indicated that such properties are related to the headgroup composition of the phospholipids (6, 7, 11, 17), the length of the acyl chains (6, 10, 15) and the degree of unsaturation of the chains (6, 10, 16). It has been shown by studies of monolayer pressurearea dependence (18), spin probe mobility and orientation (19), nmr (20) and other physical techniques that the degree of packing of lipid membranes is determined either by chain length and degree of unsaturation of the fatty acid chains of phospholipid molecules or by their specific interac-
’ Presented in part at the Brazilian Society of Pharmacology and Experimental Therapeutics (1974). N.R.C.C. No. 14211. * N.R.C.C. Visiting Scientist, 1970-73. Permanent address: Instituto de Quimica da Universidade de Sbo Paulo, C.P. 20780, Sdo Paulo, Brasil. 3 N.R.C.C. Visiting Scientist, 1971-72. Present address: Max-Planck-Institut fur biophysikalische Chemie, D-3400 Gijttingen-Nikolausberg, W. Germany. 1 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved
2
SCHREIER-MUCCILLO,
tions with other lipid molecules such as cholesterol. This in turn is related to the degree of fluidity of the hydrophobic portion of the lipid bilayer, and the rate of diffusion across the lipophilic medium will certainly be dependent on its fluidity. The permeability of solutes, both ionic (21) and nonionic (221, and of water (23) has been studied. The process of transport has been largely accounted for in terms of two fundamental steps: the partitioning of the solute from the bulk aqueous phase into the lipid medium and solute diffusion across the lipophilic portion of the bilayer (24). Some results have indicated that the rate-determining step for the permeation process consists of removal of the water of hydration of the permeant species (25, 26). We have examined the permeability properties of lipid bilayer membranes by looking at the kinetics of the reaction of the ascorbate ion with the paramagnetic group of spin probes intercalated in oriented lipid multibilayers. The method allows determination of the rate of permeation of ascorbate into rather than across the lipid bilayers. In this respect it differs from measurements on liposomes and black lipid membranes. The rate of reaction was dependent on the depth of penetration of the paramagnetic group into the bilayer and on the lipid composition of the bilayer under investigation. The net charge of the polar region of the bilayer as well as the fluidity of the lipophilic core were seen to affect the rate of the reaction. The activation energy for the process was not strongly affected by the first factor but was altered by the second. The results give further information about the location of the spin probes in the bilayer, which correlates well with the known data from the isotropic splitting factor of the probes. MATERIALS
AND
METHODS
Oriented lipid multibilayers containing spin probes were deposited on the inner, flat surfaces of standard quartz ESR aqueous sample cells by blowing wet nitrogen (high purity, oxygen free) through a chloroform solution of the lipids (27). The multibilayer films were placed under vacuum for at least 4 h to remove residual chloroform. The films were prepared from solutions which contained ca.
MARSH
AND
SMITH
10 mg/ml of phospholipid and ca. 1 mol% of spin label probe. Egg phosphatidyl choline (PC)” and bovine brain phosphatidyl serine (PSer), Grade I (indicating no dectable lipid impurities by thin layer chromatography) were obtained from Lipid Products, S. Nutfield, England, and were used without further purification. Cholesterol (cbol), recrystallized twice from methanol, was from Steraloids Inc., Pawling, N.Y. Hexadecyltrimethylammonium chloride (HDTMA) was from Eastman Organic Chemicals, Rochester, N.Y. Spin label probe I (N-2’,2’, 6’,6’-tetramethylpiperidine-1’-oxyl-stearamide) was prepared according to Hsia et al. (28); and spin label probes II, IV (Noxyl-4’,4’-dimethyloxazolidine derivatives of 5-acholestan-3-one and 5-a-androstan-3-one-17-P-01, respectively) were prepared according to Keana et al. (29). Spin label probes III, V and VI (N-oxyl-4’,4’dimethyloxazolidine derivatives of 5keto-, l2-ketoand 16-ketostearic acids, respectively) were obtained from Syva Corp., Palo Alto, Calif. Sodium ascorbate solutions were freshly prepared in 0.15 M NaCl for all systems, except when PSer was present. In the latter case a Michaelis acetate buffer (50 ml of i/7 M sodium acetate in l/7 M sodium barbital, pH adjusted with 0.1 N HCl and made up to 100 ml) was used. The pH of the NaCl/ascorbate solution was adjusted with NaOH and HCl, and work was performed in the pH 6.2-6.8 range for all systems. A Varian E-9 ESR instrument equipped for variable temperature work with a specially constructed holder to accommodate the large sample cells was used. Temperatures were measured by a thermocouple inserted next to the flat surface of the cell and are estimated to be accurate to +2.5”C. The cells containing the lipid multibilayer films were equilibrated at a given temperature and then ascorbate solution at the same temperature was added. It was observed that an instantaneous spectral change took place, indicating complete hydration of the lipid films. The kinetics of the spin probe reduction were followed by running ESR spectra of the multibilayer films, with their surface perpendicular to the applied magnetic field, at different time intervals, depending on the rate of the process. After each spectrum the field was changed by ca. 1 G (Fig. 2). The rate of decay of the height of the low field line of the spin probe spectrum was used to monitor the kinetics of the process. (This line is well outside the spectral range of the ascorbate free radical which is generated during the chemical reaction.) This criterion is justified as long as there is no change in lineshape, which is the case in all the studies performed in the present work. 4 Abbreviations used: chol, cholesterol; HDTMA, hexadecyltrimethylammonium chloride; PC, phosphatidyl choline; PSer, phosphatidyl serine.
PERMEABILITY
PROFILES
RESULTS
The reaction of ascorbate with the paramagnetic nitroxide group of water-soluble spin labels is found to take place very quickly in aqueous solution. The reaction rates for the spin probes intercalated in lipid bilayers are found to be much slower under comparable conditions. It is assumed that when the two reagents meet in the bilayer the reaction is also very rapid. The polarity of the medium is assumed not to alter the rate of reaction significantly, since it seems to be a radical process and these are not greatly affected by the polarity of the medium (47). Control experiments indicate that there was no appreciable reduction. of the ESR spectrum of spin probes in hydrated lipid bilayers to which ascorbate had not been added. The probes that were employed can be seen in Fig. 11;the relative locations of the nitroxide moieties of the various probes are also indicated; these are based both on isotropic hyperfine splitting factor meas-
k-
x-----i
‘-: + o--d-----HO’
9 HO’WV
OF LIPID
MEMBRANES
3
urements and on the present results for the rate of reduction. The systems that were studied are shown in Table I with the corresponding half-times of reaction at 19°C. Spin probe I was found to have a half-time of reaction too fast to measure at 19°C. A half-time of 3 min has been measured for probe I in PC(65%) + cho1(35%) bilayers at 3.W. In contrast, the half-time for probe II at 4°C in the same system is 13 min. Figure 2 illustrates a typical exponential decay of the spin probe ESR spectrum. A plot of the logarithm of the height of the low field line against time (Fig. 3) indicates that the rate process follows firstorder kinetics with respect to the probe concentration; the process is also first order with respect to ascorbate. A comparison between the data for the three systems in Table I, (a) HDTMA(15 mol%) + PC(50 mol%) + chol(35 mol%), (b) PC(65 mol%) + chol(35 mol%), and (c) PS(30 mol%) + PC(35 mol%) + chol(35 mol%), clearly demonstrates that the rate of the process varies in the order a > b > c for the same probe, indicating that the charges at the headgroup affect the process in the expected manner. The dependence of the reaction half-time on the type of spin probe is that expected for the relative positions of the nitroxide moieties, as indicated by the isotropic hyperfine splitting constant. A temperature variation study was per-
O-P
TABLE
I
HALF-TIMES (t,J AT 19°C M)R THE REACTION BETWEEN ASCORBATE (lo-* M, PH 6.2-6.8) ANn SPIN PROBES IN MULTIBILAYERS OF VARIOUS COMPOSITIONS
Spin probe
t,i2 bin) PC
FIG. 1. Spin labels used in this study, with diagrammatic representation of the relative ways in which they intercalate into phospholipid bilayers.
II III IV V VI ’ [Ascorbate]
9.5
16 30 32
PSer
HDTMA
KF5& + chol
(30%)
(15%)
+ PC
+ PC
(35%)
(35%)
(30%)
+ chol
+ chol
(35%)
(35%)
12 51 85 136 166
5.9" 16 34
3.2 11.5 29 60 -
= 10m3M
4
SCHREIER-MUCCILLO,
MARSH
AND
SMITH
I hn m +l
:i
FIG. 2. ESR spectra of probe II in oriented multibilayers of HDTMA(15%) + PC(50%) + cho1(35%) as a function of time after hydration with 10m3 M sodium ascorbate at pH 6.5, 6°C. The first spectrum (left) was taken immediately after hydration (~1 min) and others at regular intervals (normally, 2 min). For each measurement the starting point of the magnetic field was stepped by 1 G. Spectra were taken with the applied magnetic field perpendicular to the plane of the multibilayer film.
formed over a 20°C range (the actual range depending on the particular spin probe and being chosen such that the rate of reduction was neither too fast nor too slow to be reasonably measurable), and the activation energies for the reaction were deduced from Arrhenius plots of the halftime of reaction (Table II). This indicates that the activation energy is essentially independent of the spin probe used for a given bilayer composition and is also not changed greatly by the charge at the headgroup. The activation energy does change however when the fluidity of the system is changed by depleting the cholesterol content.
IO
0
20
30
LO
50
t (mid FIG. 3. Plots of the logarithm of the lineheights for the ESR spectra of probe II in multibilayers of PSer(30%) + PC(35%) + cho1(35%) hydrated with 10m2M sodium ascorbate, pH 6.6, 8”C, as a function of time after hydration. The three curves are for the low field (m = + l), central (m =O) and high field (m = -1) lines of the spectra taken with the magnetic field perpendicular to the plane of the multibilayer film. The zero for m = +l has been shifted to h, = 10. TABLE
II
ACTIVATION ENERGIES (E,) FOR THE REACTION BETWEEN ASCORBATE (W2 M, PH 6.2-6.81 AND SPIN PROBES IN MULTIBILAYERS OF VARIOUS COMPOSITIONS -
Spin probe
E, (kcabmol) PC (6::) + chol (35%)
II III IV V
6 6 6
14 13 16 15
PSer ‘+0;;
HDTMA
(35%) + chol (35%)
‘:“p”d (50%) + chol (35%)
15 14 -
15 16 -
DISCUSSION
A study of the permeability properties of lipid bilayers using spin label probes seems highly desirable, since this way one is actually looking at what goes on within the bilayer. The probability that the para-
magnetic groups of the probes are located at different depths in the bilayer provides a unique tool to observe the permeation process at a molecular level. The observed rates of ESR signal decay
PERMEABILITY
PROFILES
can be related to the permeability properties of the system, since it is known that the ESR signal of the nitroxide group disalmost instantaneously in appears aqueous solution or when the paramagnetic moiety is located at the polaraqueous interface of lipid bilayers (30). This observation was reinforced by the results obtained with the stearamide spin probe I in the present work. In their investigation of the headgrouplabeled phospholipid, Kornberg and McConnell (30) found that reoxidation of up to 32% of the ascorbate-reduced spin label was obtained with old preparations of egg lecithin vesicles during long incubations at 40°C. This reoxidation of the reduced label was attributed to either oxidized lipids or molecular oxygen (30) and could complicate the interpretation of the permeability profile in .the present work if an appreciable amount of the ascorbate-reduced spin label spectrum was restored by reoxidation. However, no evidence for reoxidation was seen in the first-order decays of the spin label spectrum (such as that shown in Fig. 3). Reoxidation is thought not to be important in the spin label reduction kinetics of the present study because of the following differences from the experimental situation of Kornberg and McConnell (30). (i) Freshly prepared bilayers were always used. (ii) Sonication was not used in the bilayer preparation. (iii) The time scale of the permeation experiments is much shorter than that of the Kornberg and McConnell flip-flop experiments. (iv) The permeability profile effects are evident at room temperature and below. (v) The extent of reoxidation obtained by Kornberg and McConnell during much longer incubations is small compared with the overall range of reduction effects found here for the permeability profile. Thus the observed different reduction rates for the various spin labels cannot be accounted for in terms of differential rates of reoxidation of the spin label and must be attributed to a permeation profile as the ascorbate penetrates into the bilayer. Kornberg and McConnell (30) also showed that, over a period of 20 min, ascorbate does not penetrate egg lecithin vesicles at 0°C. This was deduced from the ESR spectrum of a Tem-
OF LIPID
MEMBRANES
5
pocholine spin label occluded inside the vesicles. This result is not in contradiction with the present experiments but is simply a temperature effect: The rate of penetration of ascorbate to the centre of the bilayer is extremely slow at 0°C. The difference between the rate of reaction of the stearamide probe I and the cholestane probe II indicates that although the latter has its nitroxide group located closer to the polar-aqueous interface than any of the remaining probes III-VI, the oxazolidine ring is not in the polar headgroup region. Therefore, when spectral features are taken into account to study structural and motional properties of membrane components, it should be borne in mind that all the lipid probes used in this study, except probe I, are probing properties of the hydrophobic core of the lipid bilayer. Differences in quantitative results revealed by the use of different probes, as in a study of the effect of cholesterol on egg and dipalmitoyl phosphatidyl choline (311, are most certainly due to local differences in the hydrocarbon core and to differences in molecular shape and rigidity of the probes. We have assumed that the activation energy for the chemical reaction is not significantly changed in the lipid medium, because radical reactions are not affected much by polarity of the environment (47). Thus the kinetics of the process were analyzed as being due to permeation of ascorbate through the membrane. It has been proposed that the permeation of solutes through membranes depends on the partition coefficient of the solute between the aqueous phase and the membrane, on its diffusion constant in the membrane and on the membrane thickness (32). Our results are consistent with the first two factors. The charges on the membrane polar region repel or attract the negatively charged ascorbate ions, altering their local concentration at the headgroup region and hence their partitioning. The surface concentration of ascorbate is given by an exponential (Boltzmann) factor depending on the charge on the ascorbate ion and the surface potential of the bilayer in the ionic diffuse double layer region (see below). The effective change in
6
SCHREIER-MUCCILLO,
MARSH
AND
SMITH
, MEMBRANE BULK , activation energy on changing the headgroup charge is small-large enough to give the observed changes in the half-time C A I B of reduction but not sufficiently large to ilOW ‘24 dSC f asc n H20 ax n H20 7 affect significantly the temperature deHP pendence of the half-time, at least within the experimental accuracy of our system. I I Notice that we are comparing membranes / 1 that always contain 35 mol% cholesterol, ,( membrane 15 0 A=-6 therefore it is likely that the degree of *
PERMEABILITY
PROFILES
where [SL] denotes spin probe concentration, and the pseudo-first-order rate constant is given by k ’ = kJasc1,
where [asclo bulk aqueous the ascorbate since for the pK,, = 11.57. Hence :
III
OF ADDING CHARGED LIPIDS BILAYER SURFACE POTENTIAL
ON THE
Spin probe
PSer (30%) + PC (35%) + chol (35%) =P (-eA$/ kT)
-
11 III IV V VI Mean
[31
is the concentration in the phase and ze is the charge on ion; z = -1 at pH 6.4-6.8, ascorbate ion pK,, = 4.19,
7
MEMBRANES TABLE
EFFECT
PI
[ascl being the ascorbate concentration, assumed to remain essentially constant, and k,, the second-order rate constant. The charges and dipoles in the headgroup region of the bilayer give rise to a surface potential, +. Thus the ascorbate concentration at the bilayer surface will be given by a Bo’ltzmann factor: [ascl = [asclD.e-Zp*crm,
OF LIPID
-
A$ (mV)
HDTMA (15%) + PC (50%) + chol (35%) =P (-eA61 kT)
A$ (mV)
--
3.75 -33.2 4.44 -37.5 -27.2 2.93 -20.7 2.27 2.59 -23.9 3.20 4 -28.5 + 0.89 6.8
0.184” 0.552 0.531 0.541 k 0.014
+42.5 +14.9 +15.9 +15.4 rt_ 0.8
D Value for t1,2 corrected from [asc] = 10e3 M to [asc] = lo-’ M. These values are ignored in evaluating the mean.
constant if the model is correct. For HDTMA the values for probes IV and VI k’ = (k,,[asclO)-e+‘O’kT, 141 are similar, but the value for probe II is and since the half-time for reduction de- quite different. This latter discrepancy is duced from eq. 111 is given by tllz = probably due to the fact that this experi0.69315/k’, we get ment was performed at a much lower ascorbate concentration, in which case the kit ,,2 = (tll&-e-~*~ltT, El netics could be considerably different; this where (tIl& is the half-time of reaction for result is thus ignored in evaluating the a bilayer with zero surface potential. Equamean values in Table III. The second point tion 151 can be applied to the surface is that there is a factor of two and a change charge dependence of the reduction both of of sign between the PSer and HDTMA nitroxides located in the headgroup region values of A$. This is to be expected from a and of nitroxides situated at the same simple surface charge effect, since in the depth in the diffusive region of the bilayer, absence of clustering the 30% PSer will provided that in the latter case the diffuchange the bilayer surface charge by twice sive regions are almost identical, e.g., as much and in the opposite sign from the have the same cholesterol composition etc. 15% HDTMA. Using the fact that kT/e = The absolute value of the surface poten- 25.2 mV at 19°C and the results of Table tial is not known exactly because of uncerIII, the mean changes in bilayer surface tainties in polar headgroup orientation, potential are found to be A$(PSer) = -28.5 orientation of water dipoles etc. However, mV; A$l(HDTMA) = 15.4 mV. It perhaps the results for PC(65%) + cho1(35%) bilayshould be noted that, since these electroers may be taken as the zero level for (tl,Z>O static effects have the form of exponential in calculating the effects of PSer and factors, they should contribute to the apHDTMA on the net surface potential. The parent activation energy of the reactions. ratios tI,zl(t,,z)o = eerA*lkT will give the However, the calculated changes in activachange in surface potential on adding the tion energy are hE.(PSer) = +0.656 charged lipid. The resultant values are kcal/mol; AEJHDTMA) = -0.366 given in Table III. kcabmol, which are well within the experiThere are two main points of interest mental errors of the temperature variation arising from Table III. The first is that the experiments of Table II. values of A$ for PSer are much the same The surface potential changes that one for all spin probes, and these should be can expect to get by changing the bilayer
8 surface charge Gouy-Chapman (37)):
SCHREIER-MUCCILLO,
can be estimated from theory (see, e.g., Ref.
, MARSH
AND
SMITH
the aqueous-hydrocarbon partition coefficient, K,) suggest that this transient process takes place rather quickly and, thus, that the spin label reduction observed in is controlled by a (+ = ( ““,‘” )“’ sinh c$) 161 these experiments pseudo-steady-state diffusion. This situawhere ais the surface charge density (elec- tion would obtain if the ascorbate which removed by reactrostatic units per square centimeter), E is penetrates is instantly tion with the spin label, so that [ascl-0 in the dielectric constant of the aqueous the region of the spin label nitroxide phase (E = 80 for water), and c is the group, at least in the early stages of the concentration (moles per cubic centimeter) spin label reduction, which is the time of the electrolyte (0.15 M NaCl). Now for domain which was used in deducing the PSer, cr = (-1 et70 AZ) X 0.3, assuming a reduction rates. The concentration gramean molecular area of 70 A” in the bilayer and a net single negative charge per dient of ascorbate across the hydrophobic PSer molecule. This gives cr(PSer) = region to the nitroxide group would then constant, given by -2.06.lo4 esu/cm2 and hence, using Eq. be approximately Kg&, where Kg,, is the ascorbate concen161, the contribution to the surface potentration just inside the bilayer at the begintial from the PSer charge is A+(PSer) = ning of the diffusive region, co being the -61 mV. A similar calculation for local concentration at the surface just outHDTMA yields A$(HDTMA) = +30 mV. side the bilayer, and x is the depth at These values are to be contrasted with the which the nitroxide group is situated experimental values obtained from the within the diffusive region of the bilayer spin label reduction rates. Clearly the sur(Fig. 1). The flux of ascorbate to the spin face charge effect is of the correct order of label is then magnitude to explain the spin label reduction rates. There are several reasons why @ = 1 d_” = D K’o mol cm-2 s-1 [7] the Gouy-Chapman theory might not be A dt x completely successful in predicting the experimentally deduced changes in bilayer where co is in moles per cubic centimeter, surface potential. Changes in the dipole n is the number of moles crossing area A, contribution to the membrane potential parallel to the bilayer surface, in time t. A will probably produce significant changes more useful quantity than the flux is the in the membrane potential (37, 38). These fractional area, in the plane of the bilayer, changes will come from both the dipole swept out by the ascorbate per second. moments of the component lipids and from This is given by Q x N,, x F,,, where N,, reorientation of the water molecules associ= 6 x 10z3and F,,, is the molecular area of ated with the bilayer structure. In addithe ascorbate ion, ca. 50 A2. tion there are possible contributions from The probability of reaction between specific adsorption and discrete charge efascorbate and the spin label will be given fects arising from clustering or phase sepaby the fractional area occupied by the ration of the different lipids within the ascorbate ion multiplied by the fractional bilayer (39-41). A further discrepancy area occupied by the spin label in the could arise if there were any preferential partitioning of the spin labels in the case of plane of the bilayer. Since the spin label ESR line height, h, is proportional to the a possible lipid phase separation (42,431. spin label concentration, the rate at which 2. Diffusive Permeability Profile it decays is thus Estimates of the time taken for the ascordhldt = - @N,,F,,, x f”s&, 181 bate initially to penetrate into the bilayer (based on solution of the diffusion equawhere flL is the initial fractional area occution, with reasonable assumptions for the pied by the spin label in the plane of the value of the diffusion coefficient, D, and bilayer.
PERMEABILITY
PROFILES
Thus the pseudo-first-order rate stant is, from Eq. [ll, [7] and [a], k’ = NavcclFasc~fiI,-Wpm) and the half-time
of reduction 0.69315
t1/2=
-
MEMBRANES
con-
Ku
is x
.N,cd’,s,. f &. ’ K-P*
[lOI
As would be expected, the permeation profile for this simple assumption of uniform, constant concentration gradient is a linear pdependence of tile on 3t. To calculate orders of magnitude, we take the data for label V (1 mol%) in bilayers of PC(65 mol%) + chol(35 mol%) at 19°C: tliz = 3600 s, for c,, = 10e5 mol/cm3 (Table I). For this label x-15 A and fisL = 0.005, assuming that the spin label occupies half the area of a lipid (or cholesterol) molecule. Then K$ = 1.9*10-13 cm2/sm;unfortunately it is not possible to separate K, and D. For comparison with other experimental data, we calculate the permeability coefficient of the lipid bilayer to ascorbate:
P = (K;D/d),
OF LIPID
u11
where d is the total thickness of the diffusive region (ca. 40 A). This gives P(asc) = 4.&10-’ cm/s, which can be compared with the following values for the permeability of single bilayer, black lipid membranes to inorganic ions (44): 3.4.10-12 cm/s (K+>; lo-l2 cm/s (Na+); and 1O-g-1O-‘2 cm/s (Cl-). Thus, allowing for the more hydrophobic nature of the ascorbate ion, it seems that the above values for ascorbate may not be unreasonable in terms of this model. The very low perrneability for charged ions can be attributed partly to their low oil-water partition coefficient but also to electrostatic effects: the strong retarding image potential (9, 845)which opposes the entry of the ions into the region of low dielectric constant, the hydrocarbon region of the bilayer. This latter effect will tend to steepen and perhaps modify somewhat the diffusive permeability profile. Equation [IO] predicts a linear permeability profile. In Fig. 5 the spin label halftimes of reaction are plotted against the depth, X, at which the nitroxide group of the spin label probe is situated in the hydrophobic region of the bilaver. In deter”
x rA, FIG. 5. Permeability profile of lipid bilayers to ascorbate (lo-’ M) at WC, pH 6.2-6.8. Half-time of spin label reduction, tl, as plotted against the depth, x, at which the nitroxide is situated within the diffusive (hydrocarbon) region of the bilayer. + PC(lOO%); ( - 0 - ), PC(65mol%) (-x--A cho1(35mol%); (- A -1, PS(3Omol%) + PC(35mol%) + cho1(35mol%); (-O-j, HDTMA(15mol%) + PC(50mol%) + cho1(35mol%).
mining these depths the hydroxyl groups of probes III, IV and VI were taken as the zero point for x (see Fig. 1). A value of 1.254 h; was taken as the projected alltram C-C bond length for labels III, V and VI, and the length of the steroid nucleus for label IV was taken from Ref. (46). The position of label II was taken as x = 2.5 A, being the best fit to the profiles obtained with all the other spin-labeled probes. The permeability profiles for all the different lipid systems in Fig. 5 are reasonably linear, in accordance with Eq. [lo]; deviations from linearity could possibly be attributed to the effect of the image potential mentioned above. The linear gradient of the PC(65 mol%) + chol(35 mol%) curve of Fig. 5 yields a value of KJXPC+chol) = 1.7.10-l3 cm2/s, in agreement with the estimate made for a single spin label above. The ratio of linear gradients between the data for PC bilayers with and without cholesterol suggests that D(PC) = 1.8 X D(PC+chol), if it is assumed that the addition of cholesterol has little effect on the partition coefficient, thus quantifying the effect of cholesterol on the diffusive permeability of the bilayer. Using the ratio of linear gradients
10
SCHREIER-MUCCILLO,
(PSer+PC +chol)/(PC +chol) gives a value of A+(PSer)c -26.2 mV and similarly A$(HDTMA)f +15 mV, in agreement with the values obtained in the previous section. It should be noted, however, that the latter treatment made no assumptions about the specific form of the permeability profile, simply that the profile was unchanged on adding the charged lipid. The permeability profiles obtained in Fig. 5 give some support to the assumptions made about the relative positions of the nitroxide reporter groups of the various spin probes. In particular, the position of probe II relative to the others has been determined by the best tit of its observed reduction rates to the permeability profile. CONCLUSIONS
The penetration of ascorbate into phospholipid bilayers is found to be sensitive to both electrostatic interactions with the polar headgroups and diffusion in the hydrocarbon region. Changes in penetration rates with changing headgroup composition are attributed to changes in the bilayer surface potential which can only partly be accounted for by changes in the diffuse double layer potential. Most probably, changes in the dipolar potential (38) and possibly discrete charge effects (39) are also important. The relative insensitivity of the activation energy to headgroup composition suggests that the rate-determining step in partition is removal of water of hydration. The diffusive penetration is demonstrated by the permeation profile and the effect of cholesterol in reducing penetration rate. The diffusive mechanism can be interpreted in terms of rotational isomerism in the lipid chains (31, 34) via the Trduble “kink” theory of permeability (3). Addition of cholesterol reduces penetration rates and increases the activation energy by both decreasing the population of kinks and other isomers and increasing the rotational energy barriers for their interconversion. The permeability profile method could be readily extended to other systems, not involving spin labels, in which a permeable species is capable of reacting with a
MARSH
AND
SMITH
molecular grouping located at a specific position within the membrane. In principle this could be used to give the relative location of the specific groups, as the present study has verified the relative positions of the nitroxide groups of the various spin labels intercalated in the bilayer. Conversely, the spin label method should be immediately applicable to the study of lipid-protein interactions in biological membranes, since these may be expected to modify both the polar interactions and the diffusive permeability in the hydrocarbon phase. The present results provide useful calibration data for such experiments. ACKNOWLEDGMENTS S.S.M. is grateful to the Fundacao de Amparo a Pesquisado Estado de S&o Paulo for a fellowship during the execution of part of this work. REFERENCES 1. VAIDHYANATHAN, V. S., PERKINS, W. H. AND TOWBIN, E. J. (1964) J. Theoret. Biol. 7, 339351. 2. MANNING, G. S. (1968) J. Chen. Phys. 49,26682675. 3. TRKUBLE, H. (1971) J. Membr. Biol. 4, 193-208. 4. FISHER, R. B., AND NIMMO, I. A. (1972) J. Erp. Physiol. 57, 162-175. R. I., GARY-B• BO, C. M., AND SOL& 5. SHA’AFI, 58, 238MON, A. K. (1971) J. Gen. Physiol. 258. 6. VAN DEENEN, L. L. M. (1972) Naturwissenschaften 59, 485-491. S. G. A., SZABO, G., EISENMAN, 7. MCLAUGHLIN, G., AND CIANI, S. M. (1970) Proc. Nat. Acad. Sci. U.S.A. 67, 1268-1275. 8. LIBERMAN, YE. A., AND TOPALY, V. P. (1969) Biofiika 14, 452-461. 9. HAYDON, D. A., AND HLADKY, S. B. (1972) Quart. Rev. Biophys. 5, 187-282. 10. DE GIER, J., MANDERSLOOT, J. G., AND VAN DEENAN, L. L. M. (1968) Biochim. Biophys. Acta 150, 666-675. 11. LELIEVRE, J., AND RICH, G. T. (1973) Biochim. Biophys. Acta 298, 15-26. 12. BANGHAM, A. D. (1972)Annu. Rev. Biochem. 41, 753-776. 13. PETKAN, A., AND CHELAK, W. S. (1972) Can. J. Biochem. 50, 1030-1033. 14. BANGHAM, A. D., STANDISH, M. M., AND WATKINS, J. C. (1965) J. Mol. Biol. 13, 238-252. 15. BANGHAM, A. D., DE GIER, J., AND GREVILLE, G. D. (1967) Chem. Phys. Lipids 1, 225-246. 16. MCELHANEY, R. N., DE GIER, J., AND VAN DER
PERMEABILITY
PROFILXS
NEUT-KOU: (1973)Biochim. Biophys. Actu 298, 500-512. 17. HOPFER, U., LEHNINGER, A. L., AND LENNARZ, W. J. (197O)J. Membr. Biol. 3, 142-155. 18. DEMEL, R. A., GEURTS VAN KESSEL, W. S. M., AND VAN DEENEN, L. L. M. (1972) Biochim. Biophys. Acta 266, 26-40. 19. SMITH,
20.
21.
22. 23. 24.
25.
I. C. P.,
MCELHANEY,
26. 27. 28. 29.
SCHREIER-MUCCILLO,
S., AND
MARSH, D. (1975) in Free Radicals in Biology (Pryor, W. A., ed.), Vol. 1, Chap. 4, Academic Press, New York. CHAPMAN, D. (1972) Ann. N.Y. Acud. Sci. 195, 179-201. PAPAHADJOI’OULOS, D., AND OHKI, S. (1970) in Liquid Crystals and Ordered Fluids (Johnson, J. F., and Porter, R. S., Eds.), pp. 13-32, Plenum Press, New York. WOOD, R. E;., WIRTH, F. P., AND MORGAN, H. E. (1968)Biochim. Biophys. Actu 163,171-178. FINKELSTEIN, A., AND CASS, A. (1967) Nature (London) 216, 717-718. LIEB, W. R., AND STEIN, W. D. (1971) in Current Topics in. Membrane Transport (Bronner, F., and Kleinzeller, A., Eds.), pp. l-39, Academic Press, New York and London. DE GIER, J., MANDERSLOOT, J. G., HUPKES, J. V., R. N.,
AND
VAN
BEEK,
W.
P.
(1971)Biochim. Biophys. Actu 233,610-618. KROES, J., AND OSTWALD, R. (1971) Biochim. Biophys. A& 249, 647-650. SMITH, I. C. P. (1971) Chimiu 25, 349-360. HSIA, J. C., PIETTE, L. H., AND NOYES, R. W. (1969) J. Rep”p”. Fert. 20, 147-149. KEANA, J. F. W.e KEANA, S. B., AND BEATHAM, D. (1967) J. Am. Chem. Sot. 89,3055-3056.
R. D., 30. KORNBERG, (1971) Biochemistry
AND
MCCONNELL,
10, 1111-1120.
H.
M.
OF LIPID
MEMBRANES
11
31. SCHREIER-MUCCILLO, S., MARSH, D., DUGAS, H., SCHNEIDER, H., AND SMITH, I. C. P. (1973) Chem. Phys. Lipids 10, 11-27. 32. LIEB, W. R., AND STEIN, W. D. (1969) Nature (London) 224, 240-243. 33. GRAZIANI, Y., AND LIVNE, A. (1972) J. Membr. Biol. 7, 275-284. .34. MARSH, D. (1974) J. Membr. Biol. 18,145-162. 35. MARSH, D. (1974) Can. J. Biochem. 52,631-636. 76. TRAUBLE, H., AND HAYNES, D. H. (1971) Chem. Phys. Lipids 7, 324-335. 37. AVEYARD, R., AND HAYDON, D. A. (1973) An Introduction to the Principles of Surface Chemistry, Cambridge University Press, London. 38 HAYM)N, D. A., AND MYERS, V. B. (1973) Biochim. Biophys. Acta 307,429-443. 39. HAYNES, D. H. (1974) J. Membr. Biol. 17, 341366. 40. ~HIMSHICK, E. J., AND MCCONNELL, H. M. (1973) Biochemistry 12, 2351-2360. 41. I,ro, T., AND OHNISHI, S.-I. (1974) Biochim. Biophys. Actu 352, 29-37. 42. CLDFIELD, E., KEOUGH, K. M. AND CHAPMAN, D. (1972) FEBS Lett. 20, 344-346. 43. BUTLER, K. W., TATTRIE, N. H., AND SMITH, I. C. P. (1974) Biochim. Biophys. Actu 363,351360. 44. JAIN, M. K. (1972) The Bimolecular Lipid Membrane, Van Nostrand-Reinhold, New York. 45. GINSBERG, S., AND NOBLE, D. (1974) J. Membr. Biol. 18, 163-176. 46. VANDENHEUVEL, L. A. (1963) J. Amer. Oil Chem. Sot. 40, 455-471. 47. GOULD, E. S. (1966) Mechanism and Structure in Organic Chemistry, p. 693, Holt, Rinehart and Winston, New York.
OF BIOCHEMISTRY
Monitoring
AND BIOPHYSICS
the Permeability
S. SCHREIER-MUCCILL0,2 Division
of Biologicat
17&l-11(1976)
Profile of Lipid Membranes Probes’ D. MARSH,3
AND
Sciences, National Research Council of Canada, Received March 31, 1975
with Spin
I. C. P. SMITH Ottawa,
Canada KlA
OR6
The rate of reaction of the ascorbate ion with the nitroxide group of spin probes intercalated in lipid bilayers has been studied to examine the mechanism of transport of solutes across membranes. The loss of electron spin resonance (ESR) signal follows firstorder kinetics. For a given bilayer system, the half-time of the process increases with the distance of the reacting group from the aqueous interface, according to an approximately linear permeation profile, The dependence on phospholipid headgroup is that which would be predicted from the net charge; addition of negatively charged headgroups increases the half-time of reaction, and positively charged headgroups decrease it, compared with bilayers having no net charge. Addition of cholesterol, which is known to decrease the fluidity of the hydrocarbon core of the bilayer, is found to increase the halftime of reaction. The results have been analyzed in terms of a partition-diffusion mechanism. It is suggested that the rate-limiting step for partitioning the solute into the bilayer might be removal of water of hydration. Cholesterol increases the activation energy, most probably by increasing the height of the barriers to diffusion. Quantitation of the changes in reaction rates gives an estimate of the change in bilayer surface potential on changing the headgroup composition. Examination of the permeation profile supports a diffusive mechanism, from which it can be estimated that the diffusion coefficient is approximately halved on adding 35 mol% cholesterol to egg lecithin bilayers.
The permeability characteristics of biological membranes have been the object of much study. A large amount of theoretical work dealing with transport phenomena across membranes has been undertaken (see, e.g., Ref. (l-4)). Experimental studies have been performed both on biological and model membranes (5, 6). Electrical measurements on black lipid membranes have been used to relate such properties to permeability characteristics (7-9). Liposomes have been widely employed as model systems for the study of permeabil-
ity (10-12). In this case radioactive permeants have been used (13) as well as electrophoretic mobility data (14) and other electrical measurements (15). In a number of cases comparison has been made between permeability properties of biological membranes and of tryptosomes formed from lipid extracts of such membranes (16). The work concerned with permeability properties of lipids has indicated that such properties are related to the headgroup composition of the phospholipids (6, 7, 11, 17), the length of the acyl chains (6, 10, 15) and the degree of unsaturation of the chains (6, 10, 16). It has been shown by studies of monolayer pressurearea dependence (18), spin probe mobility and orientation (19), nmr (20) and other physical techniques that the degree of packing of lipid membranes is determined either by chain length and degree of unsaturation of the fatty acid chains of phospholipid molecules or by their specific interac-
’ Presented in part at the Brazilian Society of Pharmacology and Experimental Therapeutics (1974). N.R.C.C. No. 14211. * N.R.C.C. Visiting Scientist, 1970-73. Permanent address: Instituto de Quimica da Universidade de Sbo Paulo, C.P. 20780, Sdo Paulo, Brasil. 3 N.R.C.C. Visiting Scientist, 1971-72. Present address: Max-Planck-Institut fur biophysikalische Chemie, D-3400 Gijttingen-Nikolausberg, W. Germany. 1 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved
2
SCHREIER-MUCCILLO,
tions with other lipid molecules such as cholesterol. This in turn is related to the degree of fluidity of the hydrophobic portion of the lipid bilayer, and the rate of diffusion across the lipophilic medium will certainly be dependent on its fluidity. The permeability of solutes, both ionic (21) and nonionic (221, and of water (23) has been studied. The process of transport has been largely accounted for in terms of two fundamental steps: the partitioning of the solute from the bulk aqueous phase into the lipid medium and solute diffusion across the lipophilic portion of the bilayer (24). Some results have indicated that the rate-determining step for the permeation process consists of removal of the water of hydration of the permeant species (25, 26). We have examined the permeability properties of lipid bilayer membranes by looking at the kinetics of the reaction of the ascorbate ion with the paramagnetic group of spin probes intercalated in oriented lipid multibilayers. The method allows determination of the rate of permeation of ascorbate into rather than across the lipid bilayers. In this respect it differs from measurements on liposomes and black lipid membranes. The rate of reaction was dependent on the depth of penetration of the paramagnetic group into the bilayer and on the lipid composition of the bilayer under investigation. The net charge of the polar region of the bilayer as well as the fluidity of the lipophilic core were seen to affect the rate of the reaction. The activation energy for the process was not strongly affected by the first factor but was altered by the second. The results give further information about the location of the spin probes in the bilayer, which correlates well with the known data from the isotropic splitting factor of the probes. MATERIALS
AND
METHODS
Oriented lipid multibilayers containing spin probes were deposited on the inner, flat surfaces of standard quartz ESR aqueous sample cells by blowing wet nitrogen (high purity, oxygen free) through a chloroform solution of the lipids (27). The multibilayer films were placed under vacuum for at least 4 h to remove residual chloroform. The films were prepared from solutions which contained ca.
MARSH
AND
SMITH
10 mg/ml of phospholipid and ca. 1 mol% of spin label probe. Egg phosphatidyl choline (PC)” and bovine brain phosphatidyl serine (PSer), Grade I (indicating no dectable lipid impurities by thin layer chromatography) were obtained from Lipid Products, S. Nutfield, England, and were used without further purification. Cholesterol (cbol), recrystallized twice from methanol, was from Steraloids Inc., Pawling, N.Y. Hexadecyltrimethylammonium chloride (HDTMA) was from Eastman Organic Chemicals, Rochester, N.Y. Spin label probe I (N-2’,2’, 6’,6’-tetramethylpiperidine-1’-oxyl-stearamide) was prepared according to Hsia et al. (28); and spin label probes II, IV (Noxyl-4’,4’-dimethyloxazolidine derivatives of 5-acholestan-3-one and 5-a-androstan-3-one-17-P-01, respectively) were prepared according to Keana et al. (29). Spin label probes III, V and VI (N-oxyl-4’,4’dimethyloxazolidine derivatives of 5keto-, l2-ketoand 16-ketostearic acids, respectively) were obtained from Syva Corp., Palo Alto, Calif. Sodium ascorbate solutions were freshly prepared in 0.15 M NaCl for all systems, except when PSer was present. In the latter case a Michaelis acetate buffer (50 ml of i/7 M sodium acetate in l/7 M sodium barbital, pH adjusted with 0.1 N HCl and made up to 100 ml) was used. The pH of the NaCl/ascorbate solution was adjusted with NaOH and HCl, and work was performed in the pH 6.2-6.8 range for all systems. A Varian E-9 ESR instrument equipped for variable temperature work with a specially constructed holder to accommodate the large sample cells was used. Temperatures were measured by a thermocouple inserted next to the flat surface of the cell and are estimated to be accurate to +2.5”C. The cells containing the lipid multibilayer films were equilibrated at a given temperature and then ascorbate solution at the same temperature was added. It was observed that an instantaneous spectral change took place, indicating complete hydration of the lipid films. The kinetics of the spin probe reduction were followed by running ESR spectra of the multibilayer films, with their surface perpendicular to the applied magnetic field, at different time intervals, depending on the rate of the process. After each spectrum the field was changed by ca. 1 G (Fig. 2). The rate of decay of the height of the low field line of the spin probe spectrum was used to monitor the kinetics of the process. (This line is well outside the spectral range of the ascorbate free radical which is generated during the chemical reaction.) This criterion is justified as long as there is no change in lineshape, which is the case in all the studies performed in the present work. 4 Abbreviations used: chol, cholesterol; HDTMA, hexadecyltrimethylammonium chloride; PC, phosphatidyl choline; PSer, phosphatidyl serine.
PERMEABILITY
PROFILES
RESULTS
The reaction of ascorbate with the paramagnetic nitroxide group of water-soluble spin labels is found to take place very quickly in aqueous solution. The reaction rates for the spin probes intercalated in lipid bilayers are found to be much slower under comparable conditions. It is assumed that when the two reagents meet in the bilayer the reaction is also very rapid. The polarity of the medium is assumed not to alter the rate of reaction significantly, since it seems to be a radical process and these are not greatly affected by the polarity of the medium (47). Control experiments indicate that there was no appreciable reduction. of the ESR spectrum of spin probes in hydrated lipid bilayers to which ascorbate had not been added. The probes that were employed can be seen in Fig. 11;the relative locations of the nitroxide moieties of the various probes are also indicated; these are based both on isotropic hyperfine splitting factor meas-
k-
x-----i
‘-: + o--d-----HO’
9 HO’WV
OF LIPID
MEMBRANES
3
urements and on the present results for the rate of reduction. The systems that were studied are shown in Table I with the corresponding half-times of reaction at 19°C. Spin probe I was found to have a half-time of reaction too fast to measure at 19°C. A half-time of 3 min has been measured for probe I in PC(65%) + cho1(35%) bilayers at 3.W. In contrast, the half-time for probe II at 4°C in the same system is 13 min. Figure 2 illustrates a typical exponential decay of the spin probe ESR spectrum. A plot of the logarithm of the height of the low field line against time (Fig. 3) indicates that the rate process follows firstorder kinetics with respect to the probe concentration; the process is also first order with respect to ascorbate. A comparison between the data for the three systems in Table I, (a) HDTMA(15 mol%) + PC(50 mol%) + chol(35 mol%), (b) PC(65 mol%) + chol(35 mol%), and (c) PS(30 mol%) + PC(35 mol%) + chol(35 mol%), clearly demonstrates that the rate of the process varies in the order a > b > c for the same probe, indicating that the charges at the headgroup affect the process in the expected manner. The dependence of the reaction half-time on the type of spin probe is that expected for the relative positions of the nitroxide moieties, as indicated by the isotropic hyperfine splitting constant. A temperature variation study was per-
O-P
TABLE
I
HALF-TIMES (t,J AT 19°C M)R THE REACTION BETWEEN ASCORBATE (lo-* M, PH 6.2-6.8) ANn SPIN PROBES IN MULTIBILAYERS OF VARIOUS COMPOSITIONS
Spin probe
t,i2 bin) PC
FIG. 1. Spin labels used in this study, with diagrammatic representation of the relative ways in which they intercalate into phospholipid bilayers.
II III IV V VI ’ [Ascorbate]
9.5
16 30 32
PSer
HDTMA
KF5& + chol
(30%)
(15%)
+ PC
+ PC
(35%)
(35%)
(30%)
+ chol
+ chol
(35%)
(35%)
12 51 85 136 166
5.9" 16 34
3.2 11.5 29 60 -
= 10m3M
4
SCHREIER-MUCCILLO,
MARSH
AND
SMITH
I hn m +l
:i
FIG. 2. ESR spectra of probe II in oriented multibilayers of HDTMA(15%) + PC(50%) + cho1(35%) as a function of time after hydration with 10m3 M sodium ascorbate at pH 6.5, 6°C. The first spectrum (left) was taken immediately after hydration (~1 min) and others at regular intervals (normally, 2 min). For each measurement the starting point of the magnetic field was stepped by 1 G. Spectra were taken with the applied magnetic field perpendicular to the plane of the multibilayer film.
formed over a 20°C range (the actual range depending on the particular spin probe and being chosen such that the rate of reduction was neither too fast nor too slow to be reasonably measurable), and the activation energies for the reaction were deduced from Arrhenius plots of the halftime of reaction (Table II). This indicates that the activation energy is essentially independent of the spin probe used for a given bilayer composition and is also not changed greatly by the charge at the headgroup. The activation energy does change however when the fluidity of the system is changed by depleting the cholesterol content.
IO
0
20
30
LO
50
t (mid FIG. 3. Plots of the logarithm of the lineheights for the ESR spectra of probe II in multibilayers of PSer(30%) + PC(35%) + cho1(35%) hydrated with 10m2M sodium ascorbate, pH 6.6, 8”C, as a function of time after hydration. The three curves are for the low field (m = + l), central (m =O) and high field (m = -1) lines of the spectra taken with the magnetic field perpendicular to the plane of the multibilayer film. The zero for m = +l has been shifted to h, = 10. TABLE
II
ACTIVATION ENERGIES (E,) FOR THE REACTION BETWEEN ASCORBATE (W2 M, PH 6.2-6.81 AND SPIN PROBES IN MULTIBILAYERS OF VARIOUS COMPOSITIONS -
Spin probe
E, (kcabmol) PC (6::) + chol (35%)
II III IV V
6 6 6
14 13 16 15
PSer ‘+0;;
HDTMA
(35%) + chol (35%)
‘:“p”d (50%) + chol (35%)
15 14 -
15 16 -
DISCUSSION
A study of the permeability properties of lipid bilayers using spin label probes seems highly desirable, since this way one is actually looking at what goes on within the bilayer. The probability that the para-
magnetic groups of the probes are located at different depths in the bilayer provides a unique tool to observe the permeation process at a molecular level. The observed rates of ESR signal decay
PERMEABILITY
PROFILES
can be related to the permeability properties of the system, since it is known that the ESR signal of the nitroxide group disalmost instantaneously in appears aqueous solution or when the paramagnetic moiety is located at the polaraqueous interface of lipid bilayers (30). This observation was reinforced by the results obtained with the stearamide spin probe I in the present work. In their investigation of the headgrouplabeled phospholipid, Kornberg and McConnell (30) found that reoxidation of up to 32% of the ascorbate-reduced spin label was obtained with old preparations of egg lecithin vesicles during long incubations at 40°C. This reoxidation of the reduced label was attributed to either oxidized lipids or molecular oxygen (30) and could complicate the interpretation of the permeability profile in .the present work if an appreciable amount of the ascorbate-reduced spin label spectrum was restored by reoxidation. However, no evidence for reoxidation was seen in the first-order decays of the spin label spectrum (such as that shown in Fig. 3). Reoxidation is thought not to be important in the spin label reduction kinetics of the present study because of the following differences from the experimental situation of Kornberg and McConnell (30). (i) Freshly prepared bilayers were always used. (ii) Sonication was not used in the bilayer preparation. (iii) The time scale of the permeation experiments is much shorter than that of the Kornberg and McConnell flip-flop experiments. (iv) The permeability profile effects are evident at room temperature and below. (v) The extent of reoxidation obtained by Kornberg and McConnell during much longer incubations is small compared with the overall range of reduction effects found here for the permeability profile. Thus the observed different reduction rates for the various spin labels cannot be accounted for in terms of differential rates of reoxidation of the spin label and must be attributed to a permeation profile as the ascorbate penetrates into the bilayer. Kornberg and McConnell (30) also showed that, over a period of 20 min, ascorbate does not penetrate egg lecithin vesicles at 0°C. This was deduced from the ESR spectrum of a Tem-
OF LIPID
MEMBRANES
5
pocholine spin label occluded inside the vesicles. This result is not in contradiction with the present experiments but is simply a temperature effect: The rate of penetration of ascorbate to the centre of the bilayer is extremely slow at 0°C. The difference between the rate of reaction of the stearamide probe I and the cholestane probe II indicates that although the latter has its nitroxide group located closer to the polar-aqueous interface than any of the remaining probes III-VI, the oxazolidine ring is not in the polar headgroup region. Therefore, when spectral features are taken into account to study structural and motional properties of membrane components, it should be borne in mind that all the lipid probes used in this study, except probe I, are probing properties of the hydrophobic core of the lipid bilayer. Differences in quantitative results revealed by the use of different probes, as in a study of the effect of cholesterol on egg and dipalmitoyl phosphatidyl choline (311, are most certainly due to local differences in the hydrocarbon core and to differences in molecular shape and rigidity of the probes. We have assumed that the activation energy for the chemical reaction is not significantly changed in the lipid medium, because radical reactions are not affected much by polarity of the environment (47). Thus the kinetics of the process were analyzed as being due to permeation of ascorbate through the membrane. It has been proposed that the permeation of solutes through membranes depends on the partition coefficient of the solute between the aqueous phase and the membrane, on its diffusion constant in the membrane and on the membrane thickness (32). Our results are consistent with the first two factors. The charges on the membrane polar region repel or attract the negatively charged ascorbate ions, altering their local concentration at the headgroup region and hence their partitioning. The surface concentration of ascorbate is given by an exponential (Boltzmann) factor depending on the charge on the ascorbate ion and the surface potential of the bilayer in the ionic diffuse double layer region (see below). The effective change in
6
SCHREIER-MUCCILLO,
MARSH
AND
SMITH
, MEMBRANE BULK , activation energy on changing the headgroup charge is small-large enough to give the observed changes in the half-time C A I B of reduction but not sufficiently large to ilOW ‘24 dSC f asc n H20 ax n H20 7 affect significantly the temperature deHP pendence of the half-time, at least within the experimental accuracy of our system. I I Notice that we are comparing membranes / 1 that always contain 35 mol% cholesterol, ,( membrane 15 0 A=-6 therefore it is likely that the degree of *
PERMEABILITY
PROFILES
where [SL] denotes spin probe concentration, and the pseudo-first-order rate constant is given by k ’ = kJasc1,
where [asclo bulk aqueous the ascorbate since for the pK,, = 11.57. Hence :
III
OF ADDING CHARGED LIPIDS BILAYER SURFACE POTENTIAL
ON THE
Spin probe
PSer (30%) + PC (35%) + chol (35%) =P (-eA$/ kT)
-
11 III IV V VI Mean
[31
is the concentration in the phase and ze is the charge on ion; z = -1 at pH 6.4-6.8, ascorbate ion pK,, = 4.19,
7
MEMBRANES TABLE
EFFECT
PI
[ascl being the ascorbate concentration, assumed to remain essentially constant, and k,, the second-order rate constant. The charges and dipoles in the headgroup region of the bilayer give rise to a surface potential, +. Thus the ascorbate concentration at the bilayer surface will be given by a Bo’ltzmann factor: [ascl = [asclD.e-Zp*crm,
OF LIPID
-
A$ (mV)
HDTMA (15%) + PC (50%) + chol (35%) =P (-eA61 kT)
A$ (mV)
--
3.75 -33.2 4.44 -37.5 -27.2 2.93 -20.7 2.27 2.59 -23.9 3.20 4 -28.5 + 0.89 6.8
0.184” 0.552 0.531 0.541 k 0.014
+42.5 +14.9 +15.9 +15.4 rt_ 0.8
D Value for t1,2 corrected from [asc] = 10e3 M to [asc] = lo-’ M. These values are ignored in evaluating the mean.
constant if the model is correct. For HDTMA the values for probes IV and VI k’ = (k,,[asclO)-e+‘O’kT, 141 are similar, but the value for probe II is and since the half-time for reduction de- quite different. This latter discrepancy is duced from eq. 111 is given by tllz = probably due to the fact that this experi0.69315/k’, we get ment was performed at a much lower ascorbate concentration, in which case the kit ,,2 = (tll&-e-~*~ltT, El netics could be considerably different; this where (tIl& is the half-time of reaction for result is thus ignored in evaluating the a bilayer with zero surface potential. Equamean values in Table III. The second point tion 151 can be applied to the surface is that there is a factor of two and a change charge dependence of the reduction both of of sign between the PSer and HDTMA nitroxides located in the headgroup region values of A$. This is to be expected from a and of nitroxides situated at the same simple surface charge effect, since in the depth in the diffusive region of the bilayer, absence of clustering the 30% PSer will provided that in the latter case the diffuchange the bilayer surface charge by twice sive regions are almost identical, e.g., as much and in the opposite sign from the have the same cholesterol composition etc. 15% HDTMA. Using the fact that kT/e = The absolute value of the surface poten- 25.2 mV at 19°C and the results of Table tial is not known exactly because of uncerIII, the mean changes in bilayer surface tainties in polar headgroup orientation, potential are found to be A$(PSer) = -28.5 orientation of water dipoles etc. However, mV; A$l(HDTMA) = 15.4 mV. It perhaps the results for PC(65%) + cho1(35%) bilayshould be noted that, since these electroers may be taken as the zero level for (tl,Z>O static effects have the form of exponential in calculating the effects of PSer and factors, they should contribute to the apHDTMA on the net surface potential. The parent activation energy of the reactions. ratios tI,zl(t,,z)o = eerA*lkT will give the However, the calculated changes in activachange in surface potential on adding the tion energy are hE.(PSer) = +0.656 charged lipid. The resultant values are kcal/mol; AEJHDTMA) = -0.366 given in Table III. kcabmol, which are well within the experiThere are two main points of interest mental errors of the temperature variation arising from Table III. The first is that the experiments of Table II. values of A$ for PSer are much the same The surface potential changes that one for all spin probes, and these should be can expect to get by changing the bilayer
8 surface charge Gouy-Chapman (37)):
SCHREIER-MUCCILLO,
can be estimated from theory (see, e.g., Ref.
, MARSH
AND
SMITH
the aqueous-hydrocarbon partition coefficient, K,) suggest that this transient process takes place rather quickly and, thus, that the spin label reduction observed in is controlled by a (+ = ( ““,‘” )“’ sinh c$) 161 these experiments pseudo-steady-state diffusion. This situawhere ais the surface charge density (elec- tion would obtain if the ascorbate which removed by reactrostatic units per square centimeter), E is penetrates is instantly tion with the spin label, so that [ascl-0 in the dielectric constant of the aqueous the region of the spin label nitroxide phase (E = 80 for water), and c is the group, at least in the early stages of the concentration (moles per cubic centimeter) spin label reduction, which is the time of the electrolyte (0.15 M NaCl). Now for domain which was used in deducing the PSer, cr = (-1 et70 AZ) X 0.3, assuming a reduction rates. The concentration gramean molecular area of 70 A” in the bilayer and a net single negative charge per dient of ascorbate across the hydrophobic PSer molecule. This gives cr(PSer) = region to the nitroxide group would then constant, given by -2.06.lo4 esu/cm2 and hence, using Eq. be approximately Kg&, where Kg,, is the ascorbate concen161, the contribution to the surface potentration just inside the bilayer at the begintial from the PSer charge is A+(PSer) = ning of the diffusive region, co being the -61 mV. A similar calculation for local concentration at the surface just outHDTMA yields A$(HDTMA) = +30 mV. side the bilayer, and x is the depth at These values are to be contrasted with the which the nitroxide group is situated experimental values obtained from the within the diffusive region of the bilayer spin label reduction rates. Clearly the sur(Fig. 1). The flux of ascorbate to the spin face charge effect is of the correct order of label is then magnitude to explain the spin label reduction rates. There are several reasons why @ = 1 d_” = D K’o mol cm-2 s-1 [7] the Gouy-Chapman theory might not be A dt x completely successful in predicting the experimentally deduced changes in bilayer where co is in moles per cubic centimeter, surface potential. Changes in the dipole n is the number of moles crossing area A, contribution to the membrane potential parallel to the bilayer surface, in time t. A will probably produce significant changes more useful quantity than the flux is the in the membrane potential (37, 38). These fractional area, in the plane of the bilayer, changes will come from both the dipole swept out by the ascorbate per second. moments of the component lipids and from This is given by Q x N,, x F,,, where N,, reorientation of the water molecules associ= 6 x 10z3and F,,, is the molecular area of ated with the bilayer structure. In addithe ascorbate ion, ca. 50 A2. tion there are possible contributions from The probability of reaction between specific adsorption and discrete charge efascorbate and the spin label will be given fects arising from clustering or phase sepaby the fractional area occupied by the ration of the different lipids within the ascorbate ion multiplied by the fractional bilayer (39-41). A further discrepancy area occupied by the spin label in the could arise if there were any preferential partitioning of the spin labels in the case of plane of the bilayer. Since the spin label ESR line height, h, is proportional to the a possible lipid phase separation (42,431. spin label concentration, the rate at which 2. Diffusive Permeability Profile it decays is thus Estimates of the time taken for the ascordhldt = - @N,,F,,, x f”s&, 181 bate initially to penetrate into the bilayer (based on solution of the diffusion equawhere flL is the initial fractional area occution, with reasonable assumptions for the pied by the spin label in the plane of the value of the diffusion coefficient, D, and bilayer.
PERMEABILITY
PROFILES
Thus the pseudo-first-order rate stant is, from Eq. [ll, [7] and [a], k’ = NavcclFasc~fiI,-Wpm) and the half-time
of reduction 0.69315
t1/2=
-
MEMBRANES
con-
Ku
is x
.N,cd’,s,. f &. ’ K-P*
[lOI
As would be expected, the permeation profile for this simple assumption of uniform, constant concentration gradient is a linear pdependence of tile on 3t. To calculate orders of magnitude, we take the data for label V (1 mol%) in bilayers of PC(65 mol%) + chol(35 mol%) at 19°C: tliz = 3600 s, for c,, = 10e5 mol/cm3 (Table I). For this label x-15 A and fisL = 0.005, assuming that the spin label occupies half the area of a lipid (or cholesterol) molecule. Then K$ = 1.9*10-13 cm2/sm;unfortunately it is not possible to separate K, and D. For comparison with other experimental data, we calculate the permeability coefficient of the lipid bilayer to ascorbate:
P = (K;D/d),
OF LIPID
u11
where d is the total thickness of the diffusive region (ca. 40 A). This gives P(asc) = 4.&10-’ cm/s, which can be compared with the following values for the permeability of single bilayer, black lipid membranes to inorganic ions (44): 3.4.10-12 cm/s (K+>; lo-l2 cm/s (Na+); and 1O-g-1O-‘2 cm/s (Cl-). Thus, allowing for the more hydrophobic nature of the ascorbate ion, it seems that the above values for ascorbate may not be unreasonable in terms of this model. The very low perrneability for charged ions can be attributed partly to their low oil-water partition coefficient but also to electrostatic effects: the strong retarding image potential (9, 845)which opposes the entry of the ions into the region of low dielectric constant, the hydrocarbon region of the bilayer. This latter effect will tend to steepen and perhaps modify somewhat the diffusive permeability profile. Equation [IO] predicts a linear permeability profile. In Fig. 5 the spin label halftimes of reaction are plotted against the depth, X, at which the nitroxide group of the spin label probe is situated in the hydrophobic region of the bilaver. In deter”
x rA, FIG. 5. Permeability profile of lipid bilayers to ascorbate (lo-’ M) at WC, pH 6.2-6.8. Half-time of spin label reduction, tl, as plotted against the depth, x, at which the nitroxide is situated within the diffusive (hydrocarbon) region of the bilayer. + PC(lOO%); ( - 0 - ), PC(65mol%) (-x--A cho1(35mol%); (- A -1, PS(3Omol%) + PC(35mol%) + cho1(35mol%); (-O-j, HDTMA(15mol%) + PC(50mol%) + cho1(35mol%).
mining these depths the hydroxyl groups of probes III, IV and VI were taken as the zero point for x (see Fig. 1). A value of 1.254 h; was taken as the projected alltram C-C bond length for labels III, V and VI, and the length of the steroid nucleus for label IV was taken from Ref. (46). The position of label II was taken as x = 2.5 A, being the best fit to the profiles obtained with all the other spin-labeled probes. The permeability profiles for all the different lipid systems in Fig. 5 are reasonably linear, in accordance with Eq. [lo]; deviations from linearity could possibly be attributed to the effect of the image potential mentioned above. The linear gradient of the PC(65 mol%) + chol(35 mol%) curve of Fig. 5 yields a value of KJXPC+chol) = 1.7.10-l3 cm2/s, in agreement with the estimate made for a single spin label above. The ratio of linear gradients between the data for PC bilayers with and without cholesterol suggests that D(PC) = 1.8 X D(PC+chol), if it is assumed that the addition of cholesterol has little effect on the partition coefficient, thus quantifying the effect of cholesterol on the diffusive permeability of the bilayer. Using the ratio of linear gradients
10
SCHREIER-MUCCILLO,
(PSer+PC +chol)/(PC +chol) gives a value of A+(PSer)c -26.2 mV and similarly A$(HDTMA)f +15 mV, in agreement with the values obtained in the previous section. It should be noted, however, that the latter treatment made no assumptions about the specific form of the permeability profile, simply that the profile was unchanged on adding the charged lipid. The permeability profiles obtained in Fig. 5 give some support to the assumptions made about the relative positions of the nitroxide reporter groups of the various spin probes. In particular, the position of probe II relative to the others has been determined by the best tit of its observed reduction rates to the permeability profile. CONCLUSIONS
The penetration of ascorbate into phospholipid bilayers is found to be sensitive to both electrostatic interactions with the polar headgroups and diffusion in the hydrocarbon region. Changes in penetration rates with changing headgroup composition are attributed to changes in the bilayer surface potential which can only partly be accounted for by changes in the diffuse double layer potential. Most probably, changes in the dipolar potential (38) and possibly discrete charge effects (39) are also important. The relative insensitivity of the activation energy to headgroup composition suggests that the rate-determining step in partition is removal of water of hydration. The diffusive penetration is demonstrated by the permeation profile and the effect of cholesterol in reducing penetration rate. The diffusive mechanism can be interpreted in terms of rotational isomerism in the lipid chains (31, 34) via the Trduble “kink” theory of permeability (3). Addition of cholesterol reduces penetration rates and increases the activation energy by both decreasing the population of kinks and other isomers and increasing the rotational energy barriers for their interconversion. The permeability profile method could be readily extended to other systems, not involving spin labels, in which a permeable species is capable of reacting with a
MARSH
AND
SMITH
molecular grouping located at a specific position within the membrane. In principle this could be used to give the relative location of the specific groups, as the present study has verified the relative positions of the nitroxide groups of the various spin labels intercalated in the bilayer. Conversely, the spin label method should be immediately applicable to the study of lipid-protein interactions in biological membranes, since these may be expected to modify both the polar interactions and the diffusive permeability in the hydrocarbon phase. The present results provide useful calibration data for such experiments. ACKNOWLEDGMENTS S.S.M. is grateful to the Fundacao de Amparo a Pesquisado Estado de S&o Paulo for a fellowship during the execution of part of this work. REFERENCES 1. VAIDHYANATHAN, V. S., PERKINS, W. H. AND TOWBIN, E. J. (1964) J. Theoret. Biol. 7, 339351. 2. MANNING, G. S. (1968) J. Chen. Phys. 49,26682675. 3. TRKUBLE, H. (1971) J. Membr. Biol. 4, 193-208. 4. FISHER, R. B., AND NIMMO, I. A. (1972) J. Erp. Physiol. 57, 162-175. R. I., GARY-B• BO, C. M., AND SOL& 5. SHA’AFI, 58, 238MON, A. K. (1971) J. Gen. Physiol. 258. 6. VAN DEENEN, L. L. M. (1972) Naturwissenschaften 59, 485-491. S. G. A., SZABO, G., EISENMAN, 7. MCLAUGHLIN, G., AND CIANI, S. M. (1970) Proc. Nat. Acad. Sci. U.S.A. 67, 1268-1275. 8. LIBERMAN, YE. A., AND TOPALY, V. P. (1969) Biofiika 14, 452-461. 9. HAYDON, D. A., AND HLADKY, S. B. (1972) Quart. Rev. Biophys. 5, 187-282. 10. DE GIER, J., MANDERSLOOT, J. G., AND VAN DEENAN, L. L. M. (1968) Biochim. Biophys. Acta 150, 666-675. 11. LELIEVRE, J., AND RICH, G. T. (1973) Biochim. Biophys. Acta 298, 15-26. 12. BANGHAM, A. D. (1972)Annu. Rev. Biochem. 41, 753-776. 13. PETKAN, A., AND CHELAK, W. S. (1972) Can. J. Biochem. 50, 1030-1033. 14. BANGHAM, A. D., STANDISH, M. M., AND WATKINS, J. C. (1965) J. Mol. Biol. 13, 238-252. 15. BANGHAM, A. D., DE GIER, J., AND GREVILLE, G. D. (1967) Chem. Phys. Lipids 1, 225-246. 16. MCELHANEY, R. N., DE GIER, J., AND VAN DER
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NEUT-KOU: (1973)Biochim. Biophys. Actu 298, 500-512. 17. HOPFER, U., LEHNINGER, A. L., AND LENNARZ, W. J. (197O)J. Membr. Biol. 3, 142-155. 18. DEMEL, R. A., GEURTS VAN KESSEL, W. S. M., AND VAN DEENEN, L. L. M. (1972) Biochim. Biophys. Acta 266, 26-40. 19. SMITH,
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MARSH, D. (1975) in Free Radicals in Biology (Pryor, W. A., ed.), Vol. 1, Chap. 4, Academic Press, New York. CHAPMAN, D. (1972) Ann. N.Y. Acud. Sci. 195, 179-201. PAPAHADJOI’OULOS, D., AND OHKI, S. (1970) in Liquid Crystals and Ordered Fluids (Johnson, J. F., and Porter, R. S., Eds.), pp. 13-32, Plenum Press, New York. WOOD, R. E;., WIRTH, F. P., AND MORGAN, H. E. (1968)Biochim. Biophys. Actu 163,171-178. FINKELSTEIN, A., AND CASS, A. (1967) Nature (London) 216, 717-718. LIEB, W. R., AND STEIN, W. D. (1971) in Current Topics in. Membrane Transport (Bronner, F., and Kleinzeller, A., Eds.), pp. l-39, Academic Press, New York and London. DE GIER, J., MANDERSLOOT, J. G., HUPKES, J. V., R. N.,
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(1971)Biochim. Biophys. Actu 233,610-618. KROES, J., AND OSTWALD, R. (1971) Biochim. Biophys. A& 249, 647-650. SMITH, I. C. P. (1971) Chimiu 25, 349-360. HSIA, J. C., PIETTE, L. H., AND NOYES, R. W. (1969) J. Rep”p”. Fert. 20, 147-149. KEANA, J. F. W.e KEANA, S. B., AND BEATHAM, D. (1967) J. Am. Chem. Sot. 89,3055-3056.
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31. SCHREIER-MUCCILLO, S., MARSH, D., DUGAS, H., SCHNEIDER, H., AND SMITH, I. C. P. (1973) Chem. Phys. Lipids 10, 11-27. 32. LIEB, W. R., AND STEIN, W. D. (1969) Nature (London) 224, 240-243. 33. GRAZIANI, Y., AND LIVNE, A. (1972) J. Membr. Biol. 7, 275-284. .34. MARSH, D. (1974) J. Membr. Biol. 18,145-162. 35. MARSH, D. (1974) Can. J. Biochem. 52,631-636. 76. TRAUBLE, H., AND HAYNES, D. H. (1971) Chem. Phys. Lipids 7, 324-335. 37. AVEYARD, R., AND HAYDON, D. A. (1973) An Introduction to the Principles of Surface Chemistry, Cambridge University Press, London. 38 HAYM)N, D. A., AND MYERS, V. B. (1973) Biochim. Biophys. Acta 307,429-443. 39. HAYNES, D. H. (1974) J. Membr. Biol. 17, 341366. 40. ~HIMSHICK, E. J., AND MCCONNELL, H. M. (1973) Biochemistry 12, 2351-2360. 41. I,ro, T., AND OHNISHI, S.-I. (1974) Biochim. Biophys. Actu 352, 29-37. 42. CLDFIELD, E., KEOUGH, K. M. AND CHAPMAN, D. (1972) FEBS Lett. 20, 344-346. 43. BUTLER, K. W., TATTRIE, N. H., AND SMITH, I. C. P. (1974) Biochim. Biophys. Actu 363,351360. 44. JAIN, M. K. (1972) The Bimolecular Lipid Membrane, Van Nostrand-Reinhold, New York. 45. GINSBERG, S., AND NOBLE, D. (1974) J. Membr. Biol. 18, 163-176. 46. VANDENHEUVEL, L. A. (1963) J. Amer. Oil Chem. Sot. 40, 455-471. 47. GOULD, E. S. (1966) Mechanism and Structure in Organic Chemistry, p. 693, Holt, Rinehart and Winston, New York.