with Dipalmitoyl Lecithin - American Chemical Society

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Electrical Properties of Membrane Systems: On Mechanisms of Interaction of Ca

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with

Dipalmitoyl Lecithin at the Air-Water Interface GIUSEPPE

COLACICCO , 1

M U K U L K. BASU, and F R A N C I N E A. TANSEY

Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N Y 10461 Penetration of electrolytes into both the air-water interface and films of dipalmitoyl lecithin is accompanied by a relatively small surface potential increase, whereas hydrolysis of CaCl produces accumulation of Ca(OH) and related species at the interface (1). Although in the absence of ionic lipids a correlation between interfacial ionic populations of the electrolyte and the surface potential changes is not yet possible, the marked surface potential effects of CaCl accompanying the presence of small quantities of acidic phospholipids in dipalmitoyl lecithin films suggest that the acidic lipid contaminants are still the only certifiable species whose interaction with CaCl produces an appreciable surface potential increase. Surface radioactivity and IR absorption spectra of dipalmitoyl lecithin in the presence of CaCl produced no evidence of Ca -dipalmitoyl lecithin interaction. 2

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T n previous reports (1,2,3) we described the surface potential ( A V ) response of films of isoelectric phospholipids in the presence of anionic and cationic contaminants, either when the lipids were spread on electrolyte solution or when the latter was injected under the lipid film that had been spread on water. In the presence of 10 w t % acidic contaminant (dipalmityl phosphate, dicetyl phosphate, and dicapryl phthalate ( D C P ) 'Current address: Dr. G. Colacicco, Bioelectrochemistry Laboratory, Columbia University, BB Room 1411, 630 West 168th Street, New York, NY 10032. 0-8412-0473-X/80/33-188-057$05.00/l © 1980 American Chemical Society

58

BIOELECTROCHEMISTRY:

IONS, S U R F A C E S ,

MEMBRANES

D C P ) the A V of mixed dipalmitoyl lecithin ( D P L ) - D C P film spread on C a C l was appreciably higher than those on either N a C l or H 0 . H o w ever, when the electrolyte was injected under D P L films preformed on water, the A V remained unchanged and was thus the same on H 0 , N a C l , and C a C l ; but it increased swiftly and markedly i n the order C a C l > N a C l > H 0 when the preformed D P L film contained 10 w t % or more acidic phospholipid (1). W e interpreted this to mean that the larger A V differences on C a C l over N a C l could be attributed to the interaction of the electrolyte cation with the negative fixed charge of the diffuse double layer of the acidic contaminants. The monolayer data were consistent with the observed effects of ionic lipids onto the electrical properties of neutral phospholipids at an oil-water interface (4). W e also suggested that the A V increase of D P L films spread on C a C l as compared with films spread on H 0 could be caused by interfacial penetration of the electrolyte—penetration which would not take place when the electrolyte was injected under a preformed D P L film ( 1 ) . Although a quantitative correlation was established between the A V effect of C a and the concentration of the acidic phospholipid contaminant (1-4), no such correlation was possible between A V effect and the large quantities of electrolyte ( C a C l ) penetrated into the a i r - l i p i d - H 0 interface ( I ) . A t that time I R spectroscopy revealed a novel phenomenon of adsorption of C a i n the form of C a ( O H ) — a d s o r p t i o n which was largest at highest p H i n the absence of lipid and was still appreciable when D P L was spread on C a C l solution at p H 5.6 ( I ) . These studies bear on the much debated question whether the A V effect of G a C l is caused by interaction of C a ions w i t h the phosphate group of the neutral phospholipid. W e have maintained that such an interaction is absurd ( 1 - 6 ) , whereas the opinion is divided among various laboratories, and strangely i n the same laboratory after using two different techniques (7,8). This indicates that there is much to be clarified regarding architecture and molecular and electrostatic properties of the D P L - H 0 - e l e c t r o l y t e interface. In this chapter w e report some new data regarding: (a) the A V effect of small concentrations of acidic phospholipid; ( b ) the p H dependence of surface radioactivity i n the adsorption of C a C l ; and (c) the IR spectra of D P L films on N a C l and C a C l solutions. The purpose of this study is to obtain information and formulate new arguments toward the elucidation of molecular and electrostatic models of D P L membranes and the latter s interaction with electrolytes, with particular regard to the D P L - C a system. 2

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Materials and Methods Dipalmitoyl phosphatidyl choline ( L - a ) , alias D P L , and the sodium salt of dicetyl (hexadecyl or palmityl) phosphate were purchased from

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COLACICCO E T A L .

Electrical Properties of Membrane Systems

59

Sigma Chemical Co., St. Louis, M O . Dimyristoyl and distearoyl lecithins ( D M L and D S L ) and synthetic dipalmitoyl phosphatide acid ( D P P A ) were products of Applied Science, State College, Pa. The radiotracers C a and C1~ were obtained i n the form of C a C l and H C 1 solutions from N e w England Nuclear, Boston, M A . The methods for preparing C a C l and N a C l solutions, distilled H 0 , and the criteria for homogeneity of lipids have been described; so were the monolayer techniques for surface pressure (?r), A V , and surface radioactivity determinations as well as the procedure for the measurement of IR absorption of films and solutions by attenuated total reflectance ( A T R ) on germanium plates ( 1 , 9 ) . When necessary, details of procedures w i l l be presented with the data under the specific experiment. 4 5

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Results and Analysis Numerical values are averages of data from at least.three experiments; the deviations are presented under each figure. IR absorption curves are direct tracings from assays which were repeated at least twice. Surface Potential Studies T w o types of experiments were carried out. In one, the lipid was spread from the organic solvent onto the electrolyte solution; in the other, the lipid film was first spread on distilled H 0 and small volumes of concentrated electrolyte solution (to make the final concentration) then were injected into the aqueous phase with continuous magnetic stirring. The desired p H was obtained by adding calculated volumes of either concentrated H C 1 or N a O H solutions; the solutions were unbuffered i n order to avoid complications from the buffers ions. Films of Dipalmitoyl Lecithin and Related Lecithins Spread onto Electrolyte Solutions. I n Figure 1, the A V of spread D P L films is presented for various values of TT; D M L and D S L are included for comparison. In line with previous data (1,2,3,10,11) for the three lecithins, the A V on C a C l was higher than that on N a C l solutions, and the A V increments, A ( A V ) , became larger as the film pressure increased. In the absence of molecular correlates of surface tension and A V (1,2,5,6), we shall refrain from speculations in trying to account for the influences of the lipid structure on the A V effect of C a C l . However, since the three lecithins have the glycerylphosphoryl choline moiety i n common, the marked differences i n behavior among them (see Figure 1) must be ascribed to specific effects of the different hydrophobic chains onto several unknown lipid functions at the a i r - l i p i d - H 0 interface; among such functions we shall mention l i p i d - H 0 and l i p i d - l i p i d interactions, dielectric constant effects, the influence of the lipid on the penetration, the organization and structure of both H 0 and electrolytes, and so on. 2

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60

BIOELECTROCHEMISTRY: IONS, SURFACES, M E M B R A N E S

DML 600

Figure 1. Lipid spread on the electrolyte solution. Change of A V with the surface pressure of lecithin films spread on 150 mequiv NaCl (O) and CaCl (%), pH 5.6, 25°C: DML, DPL, and DSL. Deviations: ± 5 mV; ±0.2 dyn/cm. g

10

20

30

40

SURFACE PRESSURE ir dyne /cm

Influence of the Acidic Lipid. T o a rapid inspection (see Figure 2), the presence of the acidic D P P A ( i n mixed D P P A - D P L films spread onto the electrolyte solution) produced A V — ir curves which are similar to those in the absence of the acidic lipid. T w o features are striking. First, i n the A V — ir curves, the A V difference between C a C l (filled symbols) and N a C l (empty symbols) is nearly the same whether the concentration of acidic lipid was 10 or 50%; differential concentration effects, however, show some specificities i n the low film pressure regions (see Figure 2) and i n the A V — area curves at given film pressure values (2,3,4). Secondly, inasmuch as the appearance of the negative fixed 2

Figure 2. Lipid spread on the electrolyte solution. AV—7r curves of mixed films of DPPA and DPL spread on 150 mequiv aqueous CaCl (Curves 2 and 4) or NaCl (Curves 1 and 3), pH 5.6, 25°C. The concentrations of acidic lipid are expressed in weight percent. Deviations: ±10 mV; ±0.2 dyn/cm. DPPA: ( 10%; ( 50%. 2

SURFACE PRESSURE w dyne / cm

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COLACICCO E T A L .


61

charge of the anionic lipid at the m e m b r a n e - H 0 interface is known to produce a negative change i n A V , i.e. a negative zeta potentional (2,3,4, 6,12), the greater the concentration of the acidic lipid, the lower the A V was and, i n spite of the A V increase caused by C a over N a , the more difficult it was for the acidic mixed film to obtain the higher A V of the isoelectric lecithin. This means that at a given C a C l concentration the greater the acidic l i p i d : D P L ratio, the more the fixed negative charge remained unneutralized by C a i n the electrical diffuse double layer. Further detailed analyses must be postponed past these simple interpretations until we gain a better understanding of the molecular correlates of the surface parameters. 2

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Electrolyte Injected under the Lipid Film. There is a marked difference i n the A V response of phosphatidyl choline films to the electrolyte, depending on whether the lipid film is spread onto the electrolyte solution or the electrolyte is injected under the lecithin film that was spread first onto distilled H 0 ( I ) . Dipalmitoyl Lecithin. I n contrast to the AV—TT curves presented i n Figure 1, no difference between N a C l (empty circles) and C a C l (filled circles) is seen i n the A V — w curves at 4 m i n after the electrolyte was injected under the D P L films that had been spread on distilled H 0 at a given initial pressure (T^) value (see Figure 3 ) ; a modest increment i n A V up to 25 m V occurred between 4 and 10 min. Electrolyte injection was accompanied by small surface pressure changes about whose significance we wish not to speculate. The rise of A V w i t h the increase of film pressure (see Figure 3) is part of the general behavior of lipid films, irrespective of the composition of the aqueous phase. Influence of the Acidic Lipid. Unlike D P L films, mixed films containing 10 and 50% D P P A showed a prompt response to the introduction 2

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800 Z

r

DPL

600

LJJ

2|400 ZD to

200 20 30 10 SURFACE PRESSURE M dyne/cm

Figure 3. Electrolyte injected under DPL films. AV-rr values of DPL films at 4 min after injections of NaCl (O) or CaCl (%) in the aqueous phase (distilled H 0) to a final 150-mequiv concentration. Error as in Figure 1. %

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of C a C l into the aqueous phase. Most of the rise i n A V occurred within the first 5 min (see Figures 4 and 5). Interestingly, the magnitude of the effect is not too different i n the cases i n which the concentration of the acidic l i p i d was 10 or 50%, suggesting that saturation or most of the effect occurs at concentrations less than 10 mol % D P P A . I n a rapid inspection of the curves in Figure 4, two points of interest are noticeable. First, the initial A V values at time zero were not the same for the two concentrations of the acidic lipid, although no direct correlation is obvious. Secondly, certain features may represent specific effects to be related to structural characteristics of the acidic component, the latter's interaction with D P L i n the film, the film pressure, etc.; e.g., notice the marked difference i n the A V - t i m e curves between 10 and 50% D P P A i n the experiment i n which the initial film pressure was 10 d y n / c m . Differences were not so conspicuous for other values of the film pressure. A n y attempt at interpretation is unwarranted. A more refined study of the effect of the acidic contaminants is described i n Figure 5. In order to assess the significance of small quantities of anionic film constituents, it was important to establish the smallest concentration (of such contaminants) at which the A V effect can distinguish between C a and N a or H 0 . For an electrolyte concentration of 150 mequiv and various concentrations of D C P the A V increment of C a C l over water was related to the time which elapsed after electrolyte injection. In complete absence of exogenous acidic lipid, the kinetic curves for C a C l , N a C l , and H 0 were the same ( A ( A V ) — zero), during 2

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Figure 4. Electrolyte (150 mequiv CaCL) injected under DPL-DPPA mixed films. Kinetic curves of AV of films containing 10 wt % (upper panel) and 50 wt % (lower panel) DPPA at three different values of * (2,10, and 20 dyn/cm). Aqueous hypophase, pH 5.6, 25°C. The mixed-lipid fim at the indicated pressure was spread first on distilled H»0; the electrolyte then was injected beneath at time zero. Error as in Figure 2.

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mole % DCP

MINUTES

Figure 5. Electrolyte (150 meq CaCl ) injected under DPL-DPC mixed films. Kinetic curves of AV increase A(AVJ of mixed films of DPL containing the indicated mole percent concentration of acidic phospholipid, Na-DCP. Aqueous hypophase, pH 5.6, 25°C. The mixed films at 30 dyn/cm pressure were spread on distilled H O; the electrolyte then was injected beneath at time zero. The &(&V) values signifies the increase in AV of the DPL-DCP film on CaCL over the AV on distilled H O. By the variance analysis, the rise of the first three curves (0.1, 0.5, and 1.0 mol % DCP) over that for DPL alone was highly significant. The accuracy of the electrometer readings and the x determinations were ± 5 mV and 0.1 dyn/cm, respectively. 2

t

t

the first 4 min; but then with C a C l , the A ( A V ) value rose continuously until it reached a saturation value of 25 m V (lowest curve) at 10 min. The same lag period of 4 min was observed when 0.1 and 0.5 mol % D C P was introduced with D P L i n the mixed film; however, past that time the C a effect increased slightly though significantly above that for D P L alone. The lag time disappeared when the D C P concentrations were 1 mol % and greater; we d i d not study 0.75 mol % D C P . Inasmuch as the lag period was the same for the control and for 0.1 and 0.5 m o l % acidic lipid, the question arises as to whether the 25-mV effect of Ca** w i t h D P L is merely caused by penetration of the electrolyte into the D P L film, or is actually the effect of concentrations of acidic lipid contaminants below 0.1 mol % (contaminants which at such low levels probably cannot be removed from D P L by any known isolation procedure). A resolution of the two effects must await further experimentation. A definite message, 2

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MEMBRANES

however, is that 1 mol % acidic phospholipid is sufficient to produce a marked differentiation between C a and H 0 (see Figure 5) and N a (not shown) by the A V technique. ++

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Surface Radioactivity In various attempts at establishing the stoichiometry of an eventual interaction of C a with lecithin films, the increases i n surface radioactivity observed i n the presence of C a i n the aqueous phase were ascribed to adsorption of C a \ Apart from the interpretations, the data themselves i n this field are interestingly discordant (13,14,15). To appreciate the significance of the statement, we first present our own data from experiments in which the excess surface radioactivity i n counts per minute (A cpm) in the presence of D P L film (cpm with D P L — cpm without D P L ) are related to the hypophase p H ; the aqueous phase contained 150mM N a C l and 93/*M C a C l carrier (see Figure 6 ) . Except for a minimum at p H 3, for which we have no explanation, adsorption of C a decreased from 90 cpm at p H 1 to zero at p H 7.5; above this p H value, there was a desorption of C a , which became dramatic (200 cpm) between p H 9 and p H 10. The desorption of C a under lecithin films at high p H was observed also by Rojas and Tobias ( 1 3 ) , whereas two other groups of investigators observed a marked rise in surface radioactivity i n going from p H 4 to p H 10 (14) and p H 11 (15). Although the discrepancies among several reports are serious, they could be explained i n terms of variations i n experimental conditions, to which we referred specifically on a previous occasion (1). W i t h regard to the C a sorption i n the high p H region, we note that desorption of C a occurred from lecithin films spread onto solutions of relatively high ionic strength (150mM N a C l and 93/xAf C a C l in the present experiments, ++

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©

200 100

A CPM 0 100 ©

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Figure 6. Effect of subphase pH on adsorption and desorption of Ca*\ Films of DPL were spread on 0.15M NaCl at 25°C, containing 93/AM CaClg carrier and brought to the desired pH by addition of either HCl or NaOH solutions. The A cpm = cpm with DPL — cpm without DPL. 45

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and 5 0 m M N a C l and 100/xM C a C l in the report of Rojas and Tobias (see Ref. 13, pp. 400, 401).) Where, i n contrast, the C a adsorption increased with the p H , the ionic strength was low—namely l O m M N a C l and 0.13/xAf C a C l in one case (15) and only 100/xM C a C l in the other case (14). 2

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This account is consistent with two of our own observations. A t a given C a C l carrier concentration at p H 5.6, the C a sorption rose as the electrolyte concentration in the aqueous phase increased and it plateaued at about 150 mequiv of either N a C l or C a C l (1). In the absence of lipid, the adsorption of C a was greater as the p H of the suphase increased; this was markedly so between p H 9 and p H 10 (see Ref. 1, Table I ) . In light of the observation that, in the absence of lipid, the surge of C a at the air-water interface coincided with the accumulation of C a ( O H ) and/or other possibly related species (1), it is conceivable that with D P L films at pH's as high as 9 or 10, greater concentrations of electrolyte promote collapse of the interfacial C a ( O H ) layer and loss of C a to the aqueous phase. Such a barrier layer, however, would be stable and account for the surge of C a sorption by the lecithin films at high p H (14,15) and low ionic strength, since in the absence of lipid, C a sorption was greatest at p H 10 (1). ++

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Spectroscopic

Studies

In spite of the great confusion in data and interpretations of whether and how C a interacts with lecithins, no attempt was made to obtain information from IR spectroscopy. Using this technique, we reported that for a given concentration of C a C l i n the aqueous phase, the IR absorption by the air-water interface increased w i t h the p H of the aqueous phase, presumably from C a ( O H ) ( I ) . Furthermore, at p H 5.6 addition of DPL-spread films produced a significant increase i n the intensities of the IR absorption peaks of interfacial C a ( O H ) at 2.90, 4.65, and 6.15 /urn, and conferred to the 6.15 /Aim peak the doublet structure typical of C a ( O H ) at p H 10. In the present study we expand on the significance of the IR absorption of interfacial C a ( O H ) and we approach the question of the C a - l i p i d phosphate interaction by probing the IR absorption of D P L films on C a C l solutions at the frequencies attributed to the P = O and P - O - C bonds of the lipid phosphate at wavelengths between 8 and 10 /im. ++

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Interfacial C a ( O H ) . After either the germanium plate was dipped through the interface of the C a C l solution, or the C a C l solution was deposited onto the germanium plate, the A T R IR spectra showed three peaks at wavelengths of 2.90, 4.65, and 6.15-/xm. The peak at 4.65 /xm, which was of uncertain origin, had the smallest intensity. Since i n the 2

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MEMBRANES

absence of C a C l , there were no peaks ( i n either the absence or presence of D P L film), and since the peaks at 2.90 /xin and 6.15 fixn are attributed to O - H stretching frequencies such as those of H 0 and presumably C a ( O H ) , this I R absorption must be related either to the O - H bond of C a ( O H ) or to H 0 structures that are modified b y C a " ions and related species. The data i n Figure 7 feature two experiments—one w i t h and the other without D P L film. T h e upper panel describes the dependence of those I R absorption peaks o n the p H of the aqueous phase containing 2

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w \ /

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0 h 1

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a.

0 (0 CD < D hi a.

I

N O DPL

X(/*m) 2.90

4.65

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t z

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ii — DPL 150mM NaCl #

— DPL, 10 mM CaCI

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pH5.6 Figure 7. IR spectra (at 2.90 fim, 4.65 fim and 6.15fi m) of films collected on germanium plate from aqueous phase at 25°C, pH 5.6. Upper panel: 0.075M CaCU at different pH values without DPL films. Lower panel: DPL films spread on either 0.15M NaCl or 0.010M CaCU. The solution, with or without the lipidfilm,was left to stand at room temperature for a given time. The germanium plate then was dipped through the interface four times, dried under a stream of N$ gas, and examined in the ATR accessory (for details, see Refs. 3 and 9).

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7 5 m M C a C l i n the absence of lipid film. I n the lower panel are two curves describing the influence on the same peaks by 150mM N a C l and l O m M C a C l at p H 5.6 i n the presence of D P L films. For a given C a C l concentration, in the absence of D P L , the intensity of the I R absorption at all three frequencies increased with the p H (shown) and with the C a C l concentration at a given p H value above 4 (not shown). A feature of interest i n the upper panel is the splitting of the 6.15-/xm peak at p H 10. In the presence of D P L (lower panel), the I R absorption was n i l on 150mM N a C l and very large on l O m M C a C l , suggesting a specific effect of C a C l . Moreover, D P L on l O m M C a C l (see Figure 7) and on 7 5 m M C a C l ( I ) , p H 5.6, produced the frequency-splitting effect which was typical of C a ( O H ) at p H 10 (upper panel); the doublet d i d not appear on C a C l , p H 5.6, i n the absence of D P L (1). This suggests that at p H 5.6, D P L confers upon the interfacial H 0 or C a ( O H ) structures certain characteristics of the C a ( O H ) at p H 10. Such a spectroscopic feature might be brought about by the modifications that the lecithin operates on the interfacial lattices of the aqueous solvent. Another modification is evident also in the peak at 2.90 tun i n the form of a partial shift to lower frequencies. This can be interpreted to mean that, i n the presence of D P L (and not i n its absence, upper panel), certain populations of O - H of either H 0 or C a ( O H ) are engaged i n hydrogen bonding (18). Irrespective of whether this bonding is to other H 0 or C a ( O H ) molecules or to the polar groups of D P L , the central thesis is that it is C a that brings about such modifications i n the molecular organization of the a i r - H 0 and D P L - H 0 interfaces. Whenever C a produces effects that are demonstrable i n terms of modifications of the spectroscopic characteristics of the phosphate group of the isoelectric lecithin by either N M R (15,17) or I R (see Figure 8, Curve 3 ) , the effect does not necessarily represent direct Ca -phosphate binding, but it could mean some kinds of water binding promoted by C a . The detailed mechanisms of such phenomena remain to be established. 2

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Probing Ca -Phosphate Binding. Although this question has been at the center of the current investigations, little experimentation was reported on direct evidence which could be furnished by I R spectroscopy. In a brief mention of I R absorption of D P L - u r a n y l nitrate i n nujol paste, it was concluded that indeed C a interacts with the lipid phosphate group (7). Subsequently, after thermodynamic analysis of calorimetric studies with D P L dispersions i n aqueous electrolyte, the same laboratory suggested the absence of direct Ca -phosphate interaction (8). Strangely, the authors of the second work (8) failed to provide explanations for the discrepancy between this and a previous report (7). The particular predicament implies that either one of the two experiments had to be ++

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Figure 8. IR spectra (at 8 to 10 fim) of DPL films either deposited directly (Curve 1) or collected from the interface onto germanium plate (other curves). Curve 2, DPL on 0.15M NaCl; Curve 3, DPL on 0.001M CaCU; Curve 4, DPL on 0.010M CaCU; Curve 5, DPL on 0.075M CaCU; and Curve 6, interface of 0.075M without DPL film. Spectroscopy technique as in Figure 7.

I

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i

1 • I i 9

1 » I • 10

WAVELENGTH (X)/im

reinterpreted. I n the I R study ( 7 ) , w e see a deficiency, which w i l l be apparent during the illustration of our own work. In Figure 8 are the tracings of the I R absorption i n the 8 to 10-/*m region of the spectrum, namely, the P = O, C - O - C , and P - O - C bonds of DPL, i n films collected from the interface of aqueous solutions with different concentrations of N a C l and C a C l . F o r comparison the spectrum of D P L deposited on the germanium plate from organic solvent is presented i n Curve 1. T h e spectrum of the D P L film collected on the same plate from the surface of 150 mequiv N a C l (Curve 2) shows little modification i n the absorption of the phosphate region. W i t h I m M C a C l (Curve 3 ) , the P = 0 bond was very slightly affected (red shift from 8.00 to 8.05 fim), whereas the peak of the P - O - C stretching was shifted to a higher frequency and the slope of the absorption-wavelength curve between the P = 0 and the P - O - C frequencies was increased markedly. Both effects suggest strengthening of the P - O - C bond, probably owing to conformational and environmental changes (18) induced on the D P L molecules b y the solvent (the C a C l solution). A shift of the C - O - C frequency to greater wavelengths is also apparent, but we have no explanation for it. The shift of 5/100 fim for the P = 0 bond may be too small to be significant, but it should not be ignored, irrespective of whether the partner i n the interaction is C a itself or related C a ( O H ) and H 0 structures. More dramatic effects were observed w i t h D P L films collected from the surface of 10 and 75m Af C a C l ; the P = 0 2

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absorption peak at 8 /mm vanished, and the P - O - C peak was distorted i n l O m M C a C l and absent i n 75mAf C a C l . W e should hasten to say that disappearance of the P = 0 and P - O - C peaks cannot be interpreted to mean interaction of C a with those bonds because the IR absorption of the solvent's interface without D P L was so great (Curve 6) as to mask any specific effect of C a . The absorption blank of l O m M C a C l (not shown) bespeaks the same argument. The huge I R absorption of the C a C l blank solutions i n the 8 to 10-/xm region could be caused by the particular interfacial water structures brought about by the accumulation of C a ( O H ) at the interface. This is consistent with the increase of the 2.9 and 6.15-/*m peaks (which can be ascribed to the O - H stretching frequencies of water and possibly also of C a ( O H ) ) with increasing C a C l concentration. Masking of the 8 to 10-/um peaks of D P L by the solvent is a difficulty which was ignored by other investigators. F r o m a marked red shift of the P = 0 peak of a D P L - u r a n y l nitrate-nujol paste, Chapman et al. concluded that the uranyl ion interacted with the lecithin's phosphate group (7). Unfortunately, separate spectra of lecithin and uranyl nitrate in nujol were not shown; indeed, we could not interpret the IR spectra of D P L films collected from the surface of l O m M uranyl nitrate because a huge absorption peak of uranyl nitrate masked the P = 0 peak of lecithin 2

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Discussion and Conclusions W e have used a multiple approach in trying to establish interfacial mechanisms and structures pertaining to the interaction of C a with films of D P L . Using electrolyte injection under D P L films and A V measurements under conditions i n which C a C l has no effect (see Figure 5), we conclude beyond a doubt that acidic lipid contaminants are the only certifiable species whose interaction with C a can be identified with a prompt increase in A V . Similarly the A V effects that generally are observed when lecithin films are spread onto C a C l solutions could be produced either by the presence of undetectable quantities of anionic contaminant lipids or by the penetration and orientation of the ions of the electrolyte and water (1,5). In the light of previous experience (see Ref. 16, Figure 8), we used the injection techniques in order to gain appreciation of phenomena that could transpire from the effects of variations i n the order of addition of the interaction participants. This criterion, so well known to biochemists, should be exploited i n the study of interfacial phenomena. ++

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B y measuring surface radioactivity of D P L films spread onto aqueous solutions containing the radiotracers C a and C1", we have established 4 5

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38

70


that i n the presence of relatively high electrolyte concentration (150 mequiv N a C l (see Figure 6 ) ) , the adsorption of C a decreased from a highest value at p H 1 to zero at p H 7.5, and desorption to very large values occurred i n going from p H 7.5 to p H 10; the very large desorption of C a from D P L films at high p H is conceivable i n light of the very large adsorption of C a as C a ( O H ) at p H 10 i n the absence of D P L (see Ref. J ) , as if D P L breaks a preformed C a ( O H ) lattice, w h i c h at this p H is otherwise stable. ++

++

++

2

2

The IR studies greatly facilitated our interpretation of the data from the other studies. F o r instance, in the absence of D P L film, accumulation of C a at the interface was accompanied (a) by formation of interfacial C a ( O H ) as suggested by I R spectroscopy data and (b) by desorption of CI", as suggested by the measurements of surface radioactivity on C a C l solutions containing the C1" nuclide ( I ) . These data are consistent with a mechanism of C a C l hydrolysis; the reaction is driven to the right and thus to more C a ( O H ) accumulation i n base and to the left and thus to depletion of interfacial C a ( O H ) i n acid (see Figure 7). A concept of some biological consequence i n this respect is that although C a ( O H ) and H C 1 are products of the same hydrolysis reaction, i n the absence of D P L , C a ( O H ) accumulates at the interface whereas the constituents of HC1 seek the solution. This implies that the interfacial C a ( O H ) structures have some mechanism of defense from or inpenetrability to H C 1 . This barrier to the transport of HC1 into the lipid-free interfacial lattice could serve as an elementary model in the approach to mechanisms of membrane transport. ++

2

86

2

2

2

2

2

2

2

Relation to Other Studies. Interaction of C a w i t h phosphatidyl choline ( P C ) mono- and bimolecular membranes has been investigated carefully by Hauser and Phillips (15) and M c L a u g h l i n (17). Using a variety of approaches such as A V , surface radioactivity, zeta potential measurements, and N M R studies, both groups of investigators concluded that C a interacts with the phosphate residues of lecithin; the extent of the interaction, however, is very small since it involves less than 5% of the phosphate residues (17). ++

++

The paucity of the interaction raises new questions such as whether the interacting phosphate groups belong to the phosphatidyl choline or to some acidic lipid-phosphate contaminant, w h i c h could be silent to A V and zeta potential measurements. Such a possibility finds support i n the observation (see Figure 5) that up to 0.5 mol % , exogenous dipalmityl phosphate was not detected promptly by A V , but it was detected after several minutes. Certain techniques involving equilibration and saturation might reveal such a reservoir of acidic contaminants that are not apparent in the early phase of kinetic studies. Another possibility is the partial interaction by the hydrogen bond of the O - H groups of interfacial C a ( O H ) and specially structured H 0 w i t h the phosphate group of 2

2

4.


COLACICCO E T A L .

71

either lecithin or acidic phospholipid contaminants. This possibility finds support i n the observation of a marked though partial red shift of the 2.9 fim I R peak of either water or C a ( O H ) i n the presence of D P L films (see Figure 7, lower panel). T h e special water structures inevitably are brought about b y interfacial C a and may very well be so b y other polyvalent cations such as C o (17) w h i c h mimic or resemble C a i n their interaction with acidic lipids and interfacial water. Inasmuch as even ImAf C a C l caused a marked deformation of the I R absorption of the P - O - C bond, whereas this and the P = 0 absorptions were masked totally i n 10 and 75mAf C a C l , the question of a precise mechanism for the interaction of the lecithin-phosphate with C a must be extricated from the complexity of the viscous interfacial lattices that are formed at the C a C l concentrations used i n our experiments (19). M u c h more work is required to resolve the various molecular and ionic contributions of these membranous structures to the surface parameters such as electrical potential, surface tension, surface viscosity, and spectroscopic absorption. 2

+ +

++

++

2

2

++

2

Mechanisms and Interfacial Structures. Meanwhile, we may provide some schematic models (see Figure 9) and a critical assay of the mechanisms that reflect the views either expressed i n the literature or presented i n this communication. | — P0 " + C a

+ +

_ ^ | — P0 " C a

4

I—N

+ +

4

* I

+

r—P07

N

"0 P — i 4

+

N

^P0 -Ca 4

+ +

—I

+

-0 P^ 4

2.

+ Ca Na

+ +

2Cr-*0

+

0 Ca

JUUi era

+2NaCI

+ +

cr

cr

Figure 9. Schematic of four mechanisms describing the interaction of Ca* with films of DPL: (1) ion-dipole interaction; (2) ion-exchange mechanism; (3) ion-ion interaction with ionized anionic lipid contaminant; and (4) penetration of electrolyte, H O, and derived ions into the air-boater or the lipid-water interface. A highlight of Mechanism 4 (consistent with the surface radioactivity data, Ref. 3) is the adsorption of the ions of HCl resulting from th ehydrolysis of CaCU. The coexistence of Ca(OH), and aqueous HCl at the interface requires the formation of compartments or pools that permit the separation of the acid from the base. Such a coexistence of acidic and basic pools is conceivable in the light of the Ca(OH)t film on the HCl solution following the hydrolysis of CaCU in the absence of DLP films and is probably a characteristic of DPL films, since the adsorption of Cl~ was nil without DPL. +

t

72


According to Mechanism 1, the ion-dipole interaction proposed by Shah and Schulman (11), C a binds to the oxygen of a polarized P - O bond i n the structure of the phosphorylcholine zwitterion. I n spite of the speculative claims of Shah and Schulman (11) and the theoretical affirmations of Gillespie (20), this mechanism must be rejected on the basis of the theoretical arguments presented here and elsewhere (2,5,6). In brief, if such a bond existed (and the evidence ( I R or N M R ) is not available), it should not bear any direct relation to A V and surface dipole moments, which are generated only by ionized species and not by silent ion pairs nor b y partial charges of polarized covalent bonds ( 2 , 5 ) . ++

In Mechanism 2, which was mentioned by myself (2) and Shah and Schulman (11) and is consistent with the tenets of ion-exchange theory, C a and CI" bind respectively to the ionic phosphate and quaternary nitrogen of the unfolded salt linkage. Although consistent with the observed increase i n A V (2,6,12,21), this mechanism is contradicted b y two observations. First, i n the range between p H 4 and p H 9, the zwitterion is not titratable with either H or ' O H (22,23). Since i n the Hofmeister series H is adsorbed much more effectively than C a or Al onto acidic surfaces, it is inconceivable that C a would displace the proton at the low C a concentrations used by those who claim C a - D P L phosphate binding, unless both a special mechanism and the related energetics are accounted for fully. Secondly, the binding constant of C a with an acidic phospholipid monolayer varied with the C a concentration (15); the anomaly could be explained by large excesses of C a ions that penetrate the interface outside the binding or screening stoichiometry that is calculated from A V data, and such excesses were very large i n the case of D P L (see Figure 5, lowest curve, and Ref. 1). Finally, for the small percentage of binding (see Figure 5 and Refs. 15 and 17), one cannot discount the possibility that the binding lipid species be acidic contaminants and not lecithin itself. ++

+

+

++

+ + +

++

++

+ +

++

++

++

According to Mechanism 3, C a binds to the negative fixed charge i n the electrical double layer of an acidic lipid. Although this mechanism, proposed by myself (1,2,3,6), accounts for the observed increase i n A V , it does not account for the large quantities of C a that other approaches have shown to accumulate at the interface (see Figures 6 and 7 and Ref. 1). ++

++

Mechanism 4 also was born i n this laboratory (1-6,24). A rise in A V could be determined b y the penetration and ± : orientation of the ions of electrolyte, H 0 , and their products of interaction. Such ions could be part of either the hydration shell of the lipid's molecular organization or the thick interfacial viscosity layers that are located above and/or below the mathematical line (25) of surface tension. The measurements of I R absorption and surface radioactivity (see Figures 6,7,8 2

4.

COLACICCO E T A L .


73

and Ref. 1) provided the evidence for the complexity of the molecular composition of the interface. Detailed knowledge of the latters topography w i l l be indispensable for some understanding of the structurefunction relationship of artificial and natural membranes and w i l l be possible only by applying direct spectroscopic techniques (26) to the air-water and lipid-water interfaces i n situ, i n vivo. Acknowledgments This work was supported by Grants from the N I H Heart, L u n g , and Blood Institute. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Colacicco,G.;Basu, M. K. Biochim. Biophys. Acta 1978, 509, 230-238. Colacicco, G. Biochim. Biophys. Acta 1972, 266, 313-319. Colacicco, G. Chem. Phys. Lipids 1973,10,66-72. Colacicco, G. Nature 1965, 207, 936-938. Colacicco, G. Biophys. J. 1978,21,48a. Colacicco, G. In "Biological Horizons in Surface Science," Prince,M. L., Sears, D. F., Eds.; Academic: New York, 1973; pp. 247-284. Chapman, D.; Urbina, J.; Keough, K. M. J. Biol. Chem. 1974, 249, 25122521. Chapman, D.; Peel, W. E.; Kingston, B.; Lilley, T. H . Biochim. Biophys. Acta 1977, 464, 260-275. Colacicco, G.; Basu, M. K.; Buckelew, A. R., Jr.; Bernheimer, A. W. Biochim. Biophys. Acta 1977, 465, 378-390. Vilallonga, F.; Fernandez, M.; Rotunno, C.; Cereijido, M. Biochim. Biophys. Acta1969,183,98-109. Shah, D. O.; Schulman, J. H. J. Lipid Res. 1965, 6, 341-349. Davies, J. T.; Rideal, E. K. "Interfacial Phenomena"; Academic: New York, 1961. Rojas, E.; Tobas, J. M. Biochim. Biophys. Acta 1965, 94, 394-404. Kimizuka, H.; Kabahara, T.; Nejo, H.; Yamauchi, A. Biochim. Biophys. Acta1967,137,549-556. Hauser, H.; Philips, M. C. Proc. Intl. Congr. Surf. Act. Subst., 6th 1973, 2, 371-380. Colacicco, G. J. Colloid Interface Sci. 1969, 29, 345-364. Lau, A. L.; McLaughlin, A.; MacDonald, R.C.;McLaughlin, S. G. Chapter 3 in this book. Bellamy, L. J. "Advances in Infrared Group Frequencies"; Methuen and Co. Ltd.; Bungay, Suffolk, England, 1968. Colacicco, G.; Buckelew, A. R., Jr.; Scarpelli, E . M. J. Colloid Interface Sci. 1974,46,147-151. Gillespie, C. J. Biochim. Biophys. Acta 1970, 203, 47-61. Davies, J. T. Proc. R. Soc. 1951, A208, 224-247. Bangham, A. D.; Papahadjopoulos, D. Biochim. Biophys. Acta 1966, 126, 181-184. Abramson, M. B. J. Colloid Interface Sci. 1970, 34, 571-579. Colacicco, G.; Basu, M. K. "Abstracts of Papers, Colloid Surf. Sci. Symp.," 51st National Meeting, ACS, 1977. Adam, N. K. "The Physics and Chemistry of Surfaces"; Oxford University Press: Oxford, 1971. Ockman, N. Biochim. Biophys. Acta 1971, 345, 263-282.

RECEIVED October 17, 1978.

with Dipalmitoyl Lecithin - American Chemical Society

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