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Electrosorption of l -α-dipamitoylphosphatidylcholine at the mercury solution interface
ELEC-I’ROSORPTION OF L-cc-DIPALMITOYLPHOSPHATIDYLCHOLINE AT THE MERCURY SOLUTION INTERFACE
Department
G. T. RUNBECK, D. M. MOHILNER* and T. N. SOLIE of Chemistry, Colorado State Llniversity. Fort Collins. Colorado
80521,
USA
(Recrirrd 11 JlUW 1974)
Interfacial phenomena play an important role in biochemical and biophysical processes. The living cell is a multiphase system in which many of its functions occur at interfaces between aqueous solutions and other structures. Interfacial effects may influence the conformation of interacting molecules thereby altering the biochemical and biophysical processes [I]. The electrical double layer at the membraneesolution interface may play a marked, if not a key, role in influencing the electrical and other characteristics of the membrane. However, quantitative treatments of ion transfer processes have generally been, by necessity, restricted to arguments based on highly simplified assumptions with neglect of the double layer [Z]. To obtain physicochemically well-defined interfaces of biological signilicancc is very diflicult owing to the dynamic nature of the interfaces ill oiuo, the complexity and extreme thinness of biological membranes and the asymmetry of the media. Thus. compromises are required in the selection of interfaces in order to simplify the problem and permit analysis under carefully defined conditions. In this study the mercury-solution interface was selected in the hope that electrocapillary measurements might throw some light on the interfa: cial properties of lipids. Related work on the differential capacitance of the mercury solution interface in the presence of adsorbed liplds has been reported by Miller and Bach [3]. LXI’ERIMENTAL
The electrocapillary curves wcrc obtained using a computer controlled capillary electrometer based on the maximum-bubble-pressure principle 14, 53. The natural Bovine lecithin (“Highly Purified”) and the synthetic L-r-dipalmitoylphosphatidylcholine (A grade) were obtained from P-L Biochemicals and CALBIOCHEM, respectively. They were used without further purification. All salts were reagent grade materials and were used as received from the supplier. The water was distilled from alkaline pcrmanganate and redistilled. The methanol was twice distilled at atmospheric pressure. Gas chromatographic analysis of the purified methanol showed a single peak. lt was first attempted to measure electrocapillary curves for natural Bovine lecithin in aqueous 0.05 M sodium sulfate solutions. Various attempts to prepare * Address correspondence
to this
author.
551
Fix. I. Electrocapillary curves for natural bovine lecithin in aqueous 005 M Na,SO, at XC. Three separate experiments on the same solution are shown.
solutions of lecithin at 0.3 mg/ml, as suggested by Miller and Bach [3], for the Bovine lecithin were unsuccessful in that the electrocapillary curves were characteristic of the base electrolyte only. There was no detectable lowering of the intcrfacial tension. A “saturated solution” of the Bovine lecithin was prepared by adding excess of the solid lecithin to aqueous 0.05 M sodium sulfate followed by prolonged stirring and intermittent sonication. Chromatographic analysis on silica gel thin-layer plates showed no evidence of decomposition of the lecithin by this solubilization procedure. However, electrocapillary mcasurements on this solution gave totally irreproducible electrocapillary curves. Some examples of these curves at-e shown in Fig. I. On the other hand, differential capacitance measurements at a dropping mercury electrode using a computer controlled capacitance apparatus with a lock-in amplifier [6] gave capacitance curves qualitatively similar to those reported by Miller and Bach [3]. In a recent study of the critical micelle concentration of L-x-dipalmitoylphosphatidylcholine in water-methanol solutions, Smith and Tanford [7] have estimated the critical micelle concentration to be
552
G.
‘95
r
.... .‘I
T.
D. M.
RLJNBECK,
MOHILNER
..
..I...*..
._
SOLIE
RESULTS
.;.
‘:
-_,
.
* .
‘.
. .
-.
..‘.‘: ..,.
._
‘.._ -.
:... ,_: _.. _.
.‘,
“.. .
EMF,
T. N.
1.37(10-3)] were identical. Figure 2 shows a representative sample of the electrocapillary curves as actually measured by the computer controlled capillary electrometer.
.._.
-.. ‘._ . . . . . . . .
AND
V
Fig. 2. Rcprcsentativc electrocapillary curves for L-a-dipalmitoylphosphatidylcholine in 0.01 M NH,N03 97% (v/v) methanol-water solutions. Concentration top to bottom are: O-00, 4,4 x IO-‘, 1.7 x 10m4, 4.1 x 10e4 and I-O x lo-‘M.
4-6 x 1W lo M in water. For a natural egg lecithin dispersed in aqueous solution by ultrasonic radiation, Kelloway and Saunders [8] estimated the numberaverage micellar molecular weight to be 2 x 106. In combination these results clearly indicate that whatever is adsorbing at the mercury-solution interface for the electrocapillary and the differential capacitance measurements it is not the monomer lecithin molecules. Perhaps this accounts for the unusual electrocapillary curves which although not reproducible, exhibited a substantial lowering of the interfacial tension and change in the differential capacitance. We conclude that extreme caution is called for in the interpretation of electrosorption results obtained either electrocapillary or differential capacitance by measurements for lecithins in aqueous solutions because these solutions contain essentially only large micelles. Because of the total failure of these efforts to obtain electrocapillary curves for aqueous solutions of lecithin_ it was decided to proceed with the electrocapillary measurements, but to study the synthetic lecithin, L-a-dipalmitoylphosphatidylcholine in 97% (v/v) methanol-water solution containing OQI M NH,NO, [9]. The reference electrode was a Cl M NH&I in 95% (v/v) methanol-water calomel electrodc. A stock solution of L-a-dipalmitoylphosphatidycholine in 97% (v/v) methanol-water with O-01 M ammonium nitrate supporting electrolyte was prepared. The stock solution concentration was approximately the critical micelle concentration as estimated from the data of Smith and Tanford in the absence of electrolyte. A total of nineteen different concentrations were prepared by dilutions of the stock solution. Seventeen of these gave different electrocapillary curves. The electrocapillary curves for the three highest concentrations [lGO(10-3), 1*26(10m3),
The electrocapillary data for the solutions of the L-rA-dipalmitoylphosphatidylcholine in 97% (v/v) solutions containing 0.01 M methanol-water NH,NO, were smoothed and differentiated by digital computer using the previously published techniques [IO, 111. The charge density us potential (#-E) curves obtained from the Lippmann equation for the first 17 concentrations of the lecithin from 1.2(10)- 5M to 1+(10)-3M had a similar shape to the q”-E curves characteristic of’ many aliphatic compounds in aqueous solutions in that they did cross in a quite narrow potential range indicating the probable existence of a characteristic potential of maximum adsorption. Also the slopes of the q”-E curves in the vicinity of this potential of maximum adsorption decreased with increasing concentration indicating a lowering of the diEerentia1 capacitance with increasing lipid adsorption. However, in the case of these methanolic solutions, there was a much less drastic change in slope of the $‘-E curves in going from the lowest to the highest concentration of lecithin. This indicates that the adsorption of the lipid at the mercury-methanol interface produces a much smaller change in the differential capacitance than is characteristic of the adsorption of aliphatic compounds at the mercurywater interface. Figure 3 shows plots of the interfacial tension us logarithm of lipid concentration for seven different electrode potentials for the entire set of 19 concentrations from 1.2(10)-‘M to 1,37(10)-3M. The most unusual feature of these curves is the fact that for concentrations greater than 1.0(10)-3M there is no further lowering of the interfacial tension. This cessation of the lowering of the interfacial tension im-
Fig. 3. Representative plots of interfacial tension us logarithm of concentration of L-cr-dipalmitoylphosphatidylcholine at constant electrode potential. Electrode potentials top to bottom are: -0.50, -0.55, -060, -065, -0-70, - 0.75, - 0.80 v.
Electrosorption
plies that although the analytical concentration of lecithin in the solution is increasing, the activity of the lecithin is remaining constant for all solutions of concentration greater or equal to 1.0(10)-3M. We interpret this phenomenon to mean that at 1-0(10)-3M micelles are beginning to form. The formation of micelles is akin to the formation of the new phase, which would require that the activity of the lecithin in solution will stay constant. Once the critical micelle concentration has been reached further increase in the analytical concentration merely increases the amount of the micelle phase but the activity of the lipid and thus the interfacial tension remains constant. It appears from these data that electrocapillary measurements with the computer controlled capitlary electrometer can provide a very accurate and quite convenient method of measuring the Lritical micellc concentration of high molecular weight compounds in non-aqueous electrolyte solutions. The value, I.0f10)~3M of the critical micelle concentration of La-dipalmitoylphosphatidylcholine obtained in this study in 97% methanol-water containing 001 M NH,NO, is quite consistent with the critical micelle concentration of this same synthetic lecithin obtained by Smith and Tanford [73 in 97% methanol-water in the absence of electrolyte, namely: 16(10)-3M. The salting-out effect would be expected to lower the critrcal micelle concentration in the presence of an electrolyte. Differentiation of the curves in Fig. 3 at constant electrode potential with respect to logarithm of concentration yielded the relative surface excess, r, of the lecithin as a function of concentration and electrode potential. Figure 4 shows representative plots of r vs electrode potential for six different concentrations from 5.0(10)-5M to 6-4(10)-4M. It may be seen that the behavior is qualitatively similar to that of aliphatic compounds in aqueous solutions. In
20.0
55.3
of L-a-dipalmitoylphosphatidylcholine
r
I -40
Logarithm
-30
I -20
of concentrotim
Fig. 5. Representative adsorption isotherms at constant electrode pole&al Tar r-cc-dipalmitoylphosphatidylcholine. Electrode potentials from top to bottom arc: -O,45, -0.65, -0.85. -1.05 and +O.l5V.
particular, the adsorption maximum at each concentration does occur at approximately the same electrode potential. The adsorption isotherms at several different constant electrode potentials as a function of concentration are illustrated in Fig. 5. The most striking feature of these isotherms is the fact that there is no sign of lcvelling off up to the highest concentration, ir, the critical micelle concentration. The highest value of r observed is approximately I%( lo)- lo mole cm ’ which corresponds to an area of 92A2 occupied per molecule. Space filling molecular models indicate that for a close packed monolayer of this lecithin each molecule depending on detailed orientation, occupies an arca of approximately 4.%60AZ corresponding to r values of 3,7 2%(10- “)mole cm-‘, respectively. Evidently no plateau was obtained in the experimental adsorption isotherms because it was not possible to raise the bulk activity of the lipid to a high enough value due to the formation of mlcclles.
DISCUSSION
Electrode
potentlol.
V
Fig. 4. Representative plots of relative surface excess US electrode potential for L-a-dipalmitoylphosphatidylcholine at constant concentration. The concentration top to bottom are: 6.4 x 10e4, l-3 x 10-4, I.1 x 10-4, 9.0 X IO 5. 7.0 x lo- ’ and 5.0 x IO-’ M.
The electrocapiliary curves for L-a-dipalmitoyllecithin in 97% (v/v) methanol-water presented in this paper are generally characteristic of such curves for simpler aliphatic compounds. The derived chargedensity US potential curves suggest the existence of a potential of maximum adsorption and a lowered differential capacitance with increased lipid adsorption. The change in differential capacitance resulting from adsorption of the lipid at the mercury-methanol interface is rather smaller than that characteristically observed for other aliphatic compounds at the mercury-water interface. The adsorption isotherms presented in Fig. 5 are typical of simpler aliphatic compounds as might be expected. A noteworthy difference is that one does not get saturated coverage of the electrode with this lipid in 97% (v/v) methanolwater owing to the formation of micelles. These micelles of L-a-dipalmitoylphosphatidylcholine in 97% (v/v) methanol-water were characterized
554
G. T. Rrr~~~crc,
D. M. MOHILNER
by 220MHz proton magnetic resonance spectroscopy. Pulsed NMR measurements of the longitudinal relaxation times of the terminal methyl. the choline methyl and fatty acid chain methylene protons in the lecithin at concentrations both above and below the critical micelle concentration indicated the micelles contained only a few (2 3) monomers per micelle. This conclusion is consistent with the observations of Kcllaway and Saunders [S] regarding the size of micelles for this lipid in n-propanol-water solvents and of Elworthy and McIntosh [12] regarding the size of natural egg lecithin micclles in 93y0 (v/v) methanol-water. Thus, at the concentration where the activity of the lecithin remains constant in these experiments, the micelles that are forming contain only a few monomers per mimlle. The presence of micelles. y~‘r ‘SC, is not the explanation of the nonreproducible electrocapillary curves obtained for the sonicated aqueous lecithin solutions. However, in aqueous media the micelles formed are much larger. with molecular weights of the order of 10’ or higher. It IS not unlikely that “adsorption” of such extremely large clumps of lipid material could be the explanation for the non-reproducible lowering of the interrilclal tension such as shown in Fig. I. From these observations we conclude that double layer studies of these lipids in aqueous media are suspect. This study has shown that the original idea to study double layer properties of an adsorbed monolayer of lipid molecules on mercury in aqueous media is not reasible because of the extremely low critical micelle concentration of natural lipids in water. This study has shown that it is possible to obtain valid electrocapillary curves for lipid molecules in no*,aqueous solvents and to estimate the critical mlcelle concentration in these solvents in the prcsencc of the supporting electrolyte. However. the biological implicatlons of such non-aqueous measurements are difficult to assess. It would definitely be preferable to have measurements in aqueous solutions. We propose that a feasible approach would be to synthesize phospholipids [I31 with shorter chain fatty acids that have sufficient water solubility to permit electrocapillary measurements.
AND
T. N. SOLIE
Ackrtowlrdqrmellts-This work was supported in part by the IJS Air Force Office of Scientific Research under Grant No. AF-AFOSR-70-1887. G.T.R. acknowledges supINNI from the Biological Sciences Support Grant system (or an assistantship. T.N.S. acknowledges financial support from the College of Veterinary Medicine and Biomedical Sciences, Colorado State University. We thank Professor S. I. Chan and Dr. G. Feigenson, California Institute of Technology for making available facilities for the NMR experiments. We thank Professor Patricia R. Mohilner, Department of Computer Science, Colorado State University fol- assistance in the computer analysis of the data.
REFERENCES
Angew. Chem. 11. 551 (1972). D. E. Goldman, in Perspectives iu Menthrane Biophysics (Edited by D. P. Agin) p. 205. Gordon & Breach. New York 119721. I. R. Miller‘and-‘D. Bach. J. Colloid I!ltrrjacr Sci. 29, 250 ( 1969). J. Lawrence and D. M. Mohilner, J. rlrctrochrm. Sm. 118, 1596 (1971). J. Lawrence and D. M. Mohilner, J. electrochenr Sot. 118. 159 (19711. D. M. Mohilncr. J. C. Kreuser. H. Nakadomari and I’. R. Mohilner. EIectrochentical Society National Meering, Chicago. Illinois, May 1973. Abstract No. 202. R. Smith and C. Tanford, 1. m&c. Viol. 67, 75 (1972). I. W. Kellaway and t. Saunders. CI1em. Phps. Litds 4, 261 (1970). For a recent review of double-layer studies in nonaqueous solvents including methanol, see R. Payne, in .4dvances in Elrctrochem and Electrochvmicrcl EI2yi,zeering (Edited by P. Delahay) Vol 7. p. I. Interscience. New York (1970). D. M. Mohilner and P. R. Mobilner, .I. eiecrrorh~m. snc. 115. 261 (1968). P. R. Mohilner and D. M. Mohilner, in Comput~r.7 ill Chvrnistr~ arid Chemical Irrstrurntwtation (Edited bv .I S. Mattson, H. B. Mark, Jr and H. C. McDonald. Jr.1 Vol. 2. Elr~troclir,nichv i DD. 1-44. Marcel Dekker.
1. 5%‘. Kreutr.
2.
3. 3. 5. 6.
7. 8. 9.
IO. II.
.I
N&v
12. P. H.
York
(1972).
Elwarthy
and
D. S. McIntosh.
Kollard-2.
Z.
Department
G. T. RUNBECK, D. M. MOHILNER* and T. N. SOLIE of Chemistry, Colorado State Llniversity. Fort Collins. Colorado
80521,
USA
(Recrirrd 11 JlUW 1974)
Interfacial phenomena play an important role in biochemical and biophysical processes. The living cell is a multiphase system in which many of its functions occur at interfaces between aqueous solutions and other structures. Interfacial effects may influence the conformation of interacting molecules thereby altering the biochemical and biophysical processes [I]. The electrical double layer at the membraneesolution interface may play a marked, if not a key, role in influencing the electrical and other characteristics of the membrane. However, quantitative treatments of ion transfer processes have generally been, by necessity, restricted to arguments based on highly simplified assumptions with neglect of the double layer [Z]. To obtain physicochemically well-defined interfaces of biological signilicancc is very diflicult owing to the dynamic nature of the interfaces ill oiuo, the complexity and extreme thinness of biological membranes and the asymmetry of the media. Thus. compromises are required in the selection of interfaces in order to simplify the problem and permit analysis under carefully defined conditions. In this study the mercury-solution interface was selected in the hope that electrocapillary measurements might throw some light on the interfa: cial properties of lipids. Related work on the differential capacitance of the mercury solution interface in the presence of adsorbed liplds has been reported by Miller and Bach [3]. LXI’ERIMENTAL
The electrocapillary curves wcrc obtained using a computer controlled capillary electrometer based on the maximum-bubble-pressure principle 14, 53. The natural Bovine lecithin (“Highly Purified”) and the synthetic L-r-dipalmitoylphosphatidylcholine (A grade) were obtained from P-L Biochemicals and CALBIOCHEM, respectively. They were used without further purification. All salts were reagent grade materials and were used as received from the supplier. The water was distilled from alkaline pcrmanganate and redistilled. The methanol was twice distilled at atmospheric pressure. Gas chromatographic analysis of the purified methanol showed a single peak. lt was first attempted to measure electrocapillary curves for natural Bovine lecithin in aqueous 0.05 M sodium sulfate solutions. Various attempts to prepare * Address correspondence
to this
author.
551
Fix. I. Electrocapillary curves for natural bovine lecithin in aqueous 005 M Na,SO, at XC. Three separate experiments on the same solution are shown.
solutions of lecithin at 0.3 mg/ml, as suggested by Miller and Bach [3], for the Bovine lecithin were unsuccessful in that the electrocapillary curves were characteristic of the base electrolyte only. There was no detectable lowering of the intcrfacial tension. A “saturated solution” of the Bovine lecithin was prepared by adding excess of the solid lecithin to aqueous 0.05 M sodium sulfate followed by prolonged stirring and intermittent sonication. Chromatographic analysis on silica gel thin-layer plates showed no evidence of decomposition of the lecithin by this solubilization procedure. However, electrocapillary mcasurements on this solution gave totally irreproducible electrocapillary curves. Some examples of these curves at-e shown in Fig. I. On the other hand, differential capacitance measurements at a dropping mercury electrode using a computer controlled capacitance apparatus with a lock-in amplifier [6] gave capacitance curves qualitatively similar to those reported by Miller and Bach [3]. In a recent study of the critical micelle concentration of L-x-dipalmitoylphosphatidylcholine in water-methanol solutions, Smith and Tanford [7] have estimated the critical micelle concentration to be
552
G.
‘95
r
.... .‘I
T.
D. M.
RLJNBECK,
MOHILNER
..
..I...*..
._
SOLIE
RESULTS
.;.
‘:
-_,
.
* .
‘.
. .
-.
..‘.‘: ..,.
._
‘.._ -.
:... ,_: _.. _.
.‘,
“.. .
EMF,
T. N.
1.37(10-3)] were identical. Figure 2 shows a representative sample of the electrocapillary curves as actually measured by the computer controlled capillary electrometer.
.._.
-.. ‘._ . . . . . . . .
AND
V
Fig. 2. Rcprcsentativc electrocapillary curves for L-a-dipalmitoylphosphatidylcholine in 0.01 M NH,N03 97% (v/v) methanol-water solutions. Concentration top to bottom are: O-00, 4,4 x IO-‘, 1.7 x 10m4, 4.1 x 10e4 and I-O x lo-‘M.
4-6 x 1W lo M in water. For a natural egg lecithin dispersed in aqueous solution by ultrasonic radiation, Kelloway and Saunders [8] estimated the numberaverage micellar molecular weight to be 2 x 106. In combination these results clearly indicate that whatever is adsorbing at the mercury-solution interface for the electrocapillary and the differential capacitance measurements it is not the monomer lecithin molecules. Perhaps this accounts for the unusual electrocapillary curves which although not reproducible, exhibited a substantial lowering of the interfacial tension and change in the differential capacitance. We conclude that extreme caution is called for in the interpretation of electrosorption results obtained either electrocapillary or differential capacitance by measurements for lecithins in aqueous solutions because these solutions contain essentially only large micelles. Because of the total failure of these efforts to obtain electrocapillary curves for aqueous solutions of lecithin_ it was decided to proceed with the electrocapillary measurements, but to study the synthetic lecithin, L-a-dipalmitoylphosphatidylcholine in 97% (v/v) methanol-water solution containing OQI M NH,NO, [9]. The reference electrode was a Cl M NH&I in 95% (v/v) methanol-water calomel electrodc. A stock solution of L-a-dipalmitoylphosphatidycholine in 97% (v/v) methanol-water with O-01 M ammonium nitrate supporting electrolyte was prepared. The stock solution concentration was approximately the critical micelle concentration as estimated from the data of Smith and Tanford in the absence of electrolyte. A total of nineteen different concentrations were prepared by dilutions of the stock solution. Seventeen of these gave different electrocapillary curves. The electrocapillary curves for the three highest concentrations [lGO(10-3), 1*26(10m3),
The electrocapillary data for the solutions of the L-rA-dipalmitoylphosphatidylcholine in 97% (v/v) solutions containing 0.01 M methanol-water NH,NO, were smoothed and differentiated by digital computer using the previously published techniques [IO, 111. The charge density us potential (#-E) curves obtained from the Lippmann equation for the first 17 concentrations of the lecithin from 1.2(10)- 5M to 1+(10)-3M had a similar shape to the q”-E curves characteristic of’ many aliphatic compounds in aqueous solutions in that they did cross in a quite narrow potential range indicating the probable existence of a characteristic potential of maximum adsorption. Also the slopes of the q”-E curves in the vicinity of this potential of maximum adsorption decreased with increasing concentration indicating a lowering of the diEerentia1 capacitance with increasing lipid adsorption. However, in the case of these methanolic solutions, there was a much less drastic change in slope of the $‘-E curves in going from the lowest to the highest concentration of lecithin. This indicates that the adsorption of the lipid at the mercury-methanol interface produces a much smaller change in the differential capacitance than is characteristic of the adsorption of aliphatic compounds at the mercurywater interface. Figure 3 shows plots of the interfacial tension us logarithm of lipid concentration for seven different electrode potentials for the entire set of 19 concentrations from 1.2(10)-‘M to 1,37(10)-3M. The most unusual feature of these curves is the fact that for concentrations greater than 1.0(10)-3M there is no further lowering of the interfacial tension. This cessation of the lowering of the interfacial tension im-
Fig. 3. Representative plots of interfacial tension us logarithm of concentration of L-cr-dipalmitoylphosphatidylcholine at constant electrode potential. Electrode potentials top to bottom are: -0.50, -0.55, -060, -065, -0-70, - 0.75, - 0.80 v.
Electrosorption
plies that although the analytical concentration of lecithin in the solution is increasing, the activity of the lecithin is remaining constant for all solutions of concentration greater or equal to 1.0(10)-3M. We interpret this phenomenon to mean that at 1-0(10)-3M micelles are beginning to form. The formation of micelles is akin to the formation of the new phase, which would require that the activity of the lecithin in solution will stay constant. Once the critical micelle concentration has been reached further increase in the analytical concentration merely increases the amount of the micelle phase but the activity of the lipid and thus the interfacial tension remains constant. It appears from these data that electrocapillary measurements with the computer controlled capitlary electrometer can provide a very accurate and quite convenient method of measuring the Lritical micellc concentration of high molecular weight compounds in non-aqueous electrolyte solutions. The value, I.0f10)~3M of the critical micelle concentration of La-dipalmitoylphosphatidylcholine obtained in this study in 97% methanol-water containing 001 M NH,NO, is quite consistent with the critical micelle concentration of this same synthetic lecithin obtained by Smith and Tanford [73 in 97% methanol-water in the absence of electrolyte, namely: 16(10)-3M. The salting-out effect would be expected to lower the critrcal micelle concentration in the presence of an electrolyte. Differentiation of the curves in Fig. 3 at constant electrode potential with respect to logarithm of concentration yielded the relative surface excess, r, of the lecithin as a function of concentration and electrode potential. Figure 4 shows representative plots of r vs electrode potential for six different concentrations from 5.0(10)-5M to 6-4(10)-4M. It may be seen that the behavior is qualitatively similar to that of aliphatic compounds in aqueous solutions. In
20.0
55.3
of L-a-dipalmitoylphosphatidylcholine
r
I -40
Logarithm
-30
I -20
of concentrotim
Fig. 5. Representative adsorption isotherms at constant electrode pole&al Tar r-cc-dipalmitoylphosphatidylcholine. Electrode potentials from top to bottom arc: -O,45, -0.65, -0.85. -1.05 and +O.l5V.
particular, the adsorption maximum at each concentration does occur at approximately the same electrode potential. The adsorption isotherms at several different constant electrode potentials as a function of concentration are illustrated in Fig. 5. The most striking feature of these isotherms is the fact that there is no sign of lcvelling off up to the highest concentration, ir, the critical micelle concentration. The highest value of r observed is approximately I%( lo)- lo mole cm ’ which corresponds to an area of 92A2 occupied per molecule. Space filling molecular models indicate that for a close packed monolayer of this lecithin each molecule depending on detailed orientation, occupies an arca of approximately 4.%60AZ corresponding to r values of 3,7 2%(10- “)mole cm-‘, respectively. Evidently no plateau was obtained in the experimental adsorption isotherms because it was not possible to raise the bulk activity of the lipid to a high enough value due to the formation of mlcclles.
DISCUSSION
Electrode
potentlol.
V
Fig. 4. Representative plots of relative surface excess US electrode potential for L-a-dipalmitoylphosphatidylcholine at constant concentration. The concentration top to bottom are: 6.4 x 10e4, l-3 x 10-4, I.1 x 10-4, 9.0 X IO 5. 7.0 x lo- ’ and 5.0 x IO-’ M.
The electrocapiliary curves for L-a-dipalmitoyllecithin in 97% (v/v) methanol-water presented in this paper are generally characteristic of such curves for simpler aliphatic compounds. The derived chargedensity US potential curves suggest the existence of a potential of maximum adsorption and a lowered differential capacitance with increased lipid adsorption. The change in differential capacitance resulting from adsorption of the lipid at the mercury-methanol interface is rather smaller than that characteristically observed for other aliphatic compounds at the mercury-water interface. The adsorption isotherms presented in Fig. 5 are typical of simpler aliphatic compounds as might be expected. A noteworthy difference is that one does not get saturated coverage of the electrode with this lipid in 97% (v/v) methanolwater owing to the formation of micelles. These micelles of L-a-dipalmitoylphosphatidylcholine in 97% (v/v) methanol-water were characterized
554
G. T. Rrr~~~crc,
D. M. MOHILNER
by 220MHz proton magnetic resonance spectroscopy. Pulsed NMR measurements of the longitudinal relaxation times of the terminal methyl. the choline methyl and fatty acid chain methylene protons in the lecithin at concentrations both above and below the critical micelle concentration indicated the micelles contained only a few (2 3) monomers per micelle. This conclusion is consistent with the observations of Kcllaway and Saunders [S] regarding the size of micelles for this lipid in n-propanol-water solvents and of Elworthy and McIntosh [12] regarding the size of natural egg lecithin micclles in 93y0 (v/v) methanol-water. Thus, at the concentration where the activity of the lecithin remains constant in these experiments, the micelles that are forming contain only a few monomers per mimlle. The presence of micelles. y~‘r ‘SC, is not the explanation of the nonreproducible electrocapillary curves obtained for the sonicated aqueous lecithin solutions. However, in aqueous media the micelles formed are much larger. with molecular weights of the order of 10’ or higher. It IS not unlikely that “adsorption” of such extremely large clumps of lipid material could be the explanation for the non-reproducible lowering of the interrilclal tension such as shown in Fig. I. From these observations we conclude that double layer studies of these lipids in aqueous media are suspect. This study has shown that the original idea to study double layer properties of an adsorbed monolayer of lipid molecules on mercury in aqueous media is not reasible because of the extremely low critical micelle concentration of natural lipids in water. This study has shown that it is possible to obtain valid electrocapillary curves for lipid molecules in no*,aqueous solvents and to estimate the critical mlcelle concentration in these solvents in the prcsencc of the supporting electrolyte. However. the biological implicatlons of such non-aqueous measurements are difficult to assess. It would definitely be preferable to have measurements in aqueous solutions. We propose that a feasible approach would be to synthesize phospholipids [I31 with shorter chain fatty acids that have sufficient water solubility to permit electrocapillary measurements.
AND
T. N. SOLIE
Ackrtowlrdqrmellts-This work was supported in part by the IJS Air Force Office of Scientific Research under Grant No. AF-AFOSR-70-1887. G.T.R. acknowledges supINNI from the Biological Sciences Support Grant system (or an assistantship. T.N.S. acknowledges financial support from the College of Veterinary Medicine and Biomedical Sciences, Colorado State University. We thank Professor S. I. Chan and Dr. G. Feigenson, California Institute of Technology for making available facilities for the NMR experiments. We thank Professor Patricia R. Mohilner, Department of Computer Science, Colorado State University fol- assistance in the computer analysis of the data.
REFERENCES
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