SURFACE INTERACTION OF LECITHIN AND LYSOLECITHIN
`BY N. ROBINSON
`From the Department of Pharmaceutical Chemistry, School of Pharmacy, University
`of London, Brunswick Square, London, W.C.l
`Received June 3, 1960
`The lowering of the surface tension of water by lysolecithin in the
`presence of a small constant amount of lecithin has been investigated
`to examine the changes in the boundary tension during the formation
`of a primary phosphatide membrane.
`In the higher concentration
`range of lysolecithin (above 0.05 per cent w/v corresponding to a
`weight fraction of 0.4) the surface activity was not affected by the
`presence of lecithin. Below 0.05 per cent w/v lysolecithin the surface
`activity was reduced but in the very dilute region of the critical micelle
`concentration it was restored.
`The presence of calcium chloride reduced the surface activity to a
`greater extent above the critical micelle concentration region of lysole-
`cithin than below. The effects of potassium chloride differed from
`calcium chloride for different regions of the lysolecithin concentration
`range. Ageing effects due to the salts affected the surface acitivity,
`probably by reason of the removal of lecithin from the interface.
`The results were complex and only a qualitative interpretation of the
`behaviour was attempted. The surface
`tension : concentration
`relation for aqueous lecithin sols at four different temperatures, a
`precursor to the main work, showed a lowering of the surface tension
`of water to less than 41 dyne/cm. by 0.5 per cent w/v lecithin at 25" ;
`reducing the concentration to 0.05 per cent w/v the surface activity of
`lecithin steadily diminished to zero.
`SINCE lysolecithin is an enzymatic breakdown product of lecithin, these
`two substances occur together in biological systems and the surface
`properties of one will be modified by the presence of the other.
`In 1957 Elworthy and Saundersl suggested that when stable boundaries
`were formed between an aqueous phosphatide sol and water, the structure
`of the interfacial film was that of an extended bimolecular leaflet, with
`polar groups on its outside surface. This concept bore some resemblance
`to Danielli and Davson's2 general structure of a simple cell membrane.
`Later Saunders3 observed that lysolecithin and lecithin interacted, when
`present in certain proportions, to form highly viscous sols. He suggested
`that if sufficient lysolecithin was present in the internal fluid of a cell the
`lecithin present would be stable to monovalent metal ions, but precipita-
`tion of a phosphatide membrane could still occur when the internal
`fluid met a solution containing divalent metal ions. At the weight fraction
`necessary to give precipitation the system was not lytic and hence the
`membrane would be stable. Robinson and Saunders4 have reported
`that the strong interaction of lysolecithin and monostearin to form a
`viscous sol may also be indicative of typical lysolecithin-lipid cohesive
`forces contributory to the rigidity of a cell membrane.
`The strength of the membrane will be governed in some measure by
`the change in surface tension of the membrane boundary according to the
`amount of lysolecithin present within the internal fluid. The latter will,
`609
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`IPR2018-00080
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`N. ROBINSON
`in turn, be influenced by the concentration of lecithinase catalysing the
`hydrolysis of lecithin, hydrogen ion concentration, ionic strength and
`other environmental conditions. The surface interaction of lysolecithin
`and lecithin has been studied to determine the extent of modification of
`a boundary tension by changes in some of these conditions.
`Both phosphatides possess surface activities to different extents and the
`lowering of the surface tension of water when both components are
`present will depend upon interaction in solution. Lysolecithin could exert
`a solubilising power on lecithin thus tending to remove lecithin from the
`interface. It is suggested that the physical state of the mixed phos-
`phatide aggregate in the bulk phase will not be one of lecithin solubilised
`within the lipophilic region of the lysolecithin micelle in the conventional
`manner ; this is prevented by the hydrophilic phosphoryl choline head-
`group of the lecithin molecule. It is more probable that the surface of a
`lysolecithin micelle will be impregnated with single lecithin molecules.
`The physical state of this mixed phosphatide aggregate will be reported
`later.
`Lecithin sols are sensitive to very small amounts of sodium, potassium
`and calcium chlorides and the presence of these salts was expected to
`modify the surface activity of lecithin sols. Lysolecithin sols are stable
`to small amounts of electrolytes and the surface tension of these sols is
`comparatively unaffected by their presence.
`In studying the surface
`effects of sols containing both phosphatide components, a concentration
`of lecithin was chosen sufficient to influence the behaviour of the lysole-
`cithin component whilst independently lecithin exerted little or no surface
`activity itself. An initial study of the lowering of the surface tension of
`water by lecithin showed that at a concentration of 0.05 per cent w/v
`lecithin its surface activity was negligible. This concentration was there-
`for chosen for the work.
`The measurements in these experiments were taken one hour after
`preparation of the sols, but systems containing salts were re-examined
`after 24 and 96 hours.
`
`EXPERIMENTAL
`Preparation of Lecithin and Lysolecithin
`Lecithin was prepared from egg yolks as previously described4.
`Lysolecithin was prepared from a sample of the lecithin using Russell
`viper venom according to Saunders3 modification of the method of
`Hanahan5. Analytical figures for the two substances are given below.
`Lecithin
`Lysolecithin
`. .
`1.75
`2.72
`Nitrogen (per cent)
`. . 3.89
`5.98
`Phosphorus (per cent)
`. . 0.99: 1
`N : P ratio . .
`..
`1 : 1.02
`. . 72
`. .
`4.2
`Iodine number
`Preparation of lecithin sols. Lecithin sols were prepared by evaporating
`a sample of the stock solution of lecithin to dryness, dissolving the
`weighed quantity of lecithin in a minimum volume of ether, adding
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`SURFACE INTERACTION OF LECITHIN AND LYSOLECITHIN
`distilled water and evaporating the ether with a stream of nitrogen. The
`sols were freed from air on a filter pump, passed down a small column of
`mixed strong ion exchange resins and made up to volume.
`The mixed sols were prepared by taking
`Preparation of mixed sols.
`measured quantities of stock solutions of the two phosphatides of known
`concentrations, mixing and evaporating to dryness. The film of intimately
`
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`36
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`0.4
`0.6
`0.2
`Concentration of lecithin (per cent w/v)
`FIG. 1. Variation of surface tension of water with concentration of lecithin at
`xZO", O W , U32.5", A40".
`mixed phosphatides was dispersed in warm distilled water and shaken to
`give a clear sol. Traces of electrolytes and dissolved air were removed.
`Sols containing potassium chloride and calcium chloride were prepared
`as previously and made up to volume by addition of small calculated
`volumes of concentrated salt solutions.
`Surface tensions between 20" and 40" were measured by
`Apparatus.
`the ring method using the chainomatic balance assembly previously
`described6.
`
`RESULTS AND DISCUSSION
`Surface Tension Eflects of Lecithin
`The surface activity of lecithin at a concentration of 0.5 per cent wjv
`was marked, the surface tension of water being lowered to less than
`41 dyne/cm. at the four temperatures investigated (Fig. 1). Smaller
`concentrations continued to produce a considerable lowering of the
`61 1
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`

`N. ROBINSON
`surface tension but at 0.05 per cent w/v the effect became negligible except
`at 40" when the surface tension was 66-6 dynelcm. The results did not
`show any abrupt change in the surface tension : concentration relationship
`indicating that the critical micelle concentration of lecithin in water was
`very low ; the good balance between the hydrophilic and lipophilic groups
`in the lecithin molecule suggests that aggregates would be present below
`the concentrations range examined.
`The surface tension effects of lecithin on water under different condi-
`tions have been previously reported, but more recent preparations of
`
`68
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`0.002
`
`0.01
`
`0.05
`
`36
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`0
`
`0.004
`0.006
`0.008
`Concentration of lysolecithin (per cent w/v)
`FIG. 2.
`ERect of CaC1, on thesurface tension of mixed lysolecithin-lecithin sols at 25".
`x Lysolecithin
`0 Mixed sols of lysolecithin and 0.05 per cent w/v lecithin
`0 Mixed sols in
`CaC1,
`A Mixed sols in
`CaC1, after 24 hours
`0 Mixed sols in
`CaCI, after 96 hours
`lecithin by chromatography indicated that small amounts of lysolecithin
`and other phosphatides were probably p r e ~ e n t ~ - ~ . An equally successful
`but more rapid method for the final purification of lecithin using ion
`exchange resins has been reported by Perrin and SaunderslO. The high
`surface activity of lysolecithin could greatly affect measurements of the
`surface tension of aqueous lecithin sols.
`Earlier studies by Neuschlozll, using a drop number method, showed
`that salts brought about a change in the lowering effects of lecithin on the
`surface tension of water, aluminium chloride inhibiting the surface
`activity in smaller concentrations than calcium, sodium and potassium
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`SURFACE INTERACTION OF LECITHIN AND LYSOLECITHIN
`chlorides. Price and LewiP, using the maximum bubble pressure
`method, obtained a maximum in the surface tension : concentration
`relationship at pH 2-6 which was thought to be the isoelectric point.
`Fischgold and Chain13 have since shown that the isoelectric point is in
`fact much higher (6.7). The experiments of Boutaric and Berthier14
`showed a lowering of the surface tension of water by 0.5 per cent wjv
`
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`0.002
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`0.01
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`0.05
`
`0.008
`0.006
`0.004
`Concentration of lysolecithin (per cent w/v)
`FIG. 3. Effect of CaCI, on the surface tension of mixed lysolecithin-lecithin sols at
`For key see Fig. 2.
`40".
`lecithin to 32-6 dyne/cm. after 1 hour; the effect of salts on the surface
`tension of lecithin sols was, however, contrary to results obtained by
`previous workers.
`Surface Interaction with Lecithin
`Throughout the concentration range of lysolecithin at 25" its surface
`activity was depressed to different extents by the presence of lecithin.
`Above a concentration of 0.05 per cent w/v, sufficient lysolecithin was
`present to remove lecithin from the interface and the surface tension of the
`sol remained relatively unaltered. Below this concentratin the rise in
`surface tension suggested that the lecithin brought about a withdrawal
`of lysolecithin away from the air-water interface to participate in the
`In this concentration region the boundary
`solubilisation of the lecithin.
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`

`N. ROBINSON
`tension of a membrane could be lessened by removal of lecithin from
`the interface resulting in instability which may lead to some lysing action.
`At a concentration approaching the critical micelle concentration of
`lysolecithin (mol ratio of lysolecithin to lecithin approximately 1 : 20) a
`marked increase in surface activity took place which was attributed to
`small aggregates and single molecules, which possessed little solubilising
`
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`36
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`0.004
`0.006
`Concentration of lysolecithin (per cent w/v)
`FIG. 4.
`Effect of KCl on the surface tension of mixed lysolecithin-lecithin sols at 25".
`X Lysolecithin
`0 Mixed sols of lysolecithin and 0.25 per cent w/v lecithin
`0 Mixed sols in ~O-*M KC1
`0 Mixed sols in 10-IM KCI
`A Mixed sols in 10-'M KCl after 24 hours
`C ) Mixed sols in 1 0 - I ~ KCl after 96 hours
`power, present at the interface. The surface tension at high dilution was
`slightly less than that of a pure lysolecithin sol.
`At 40" a similar lowering of the surface tension of water took place
`except below the region of 0401 per cent w/v. The plateau in this region
`contrasted strongly with the behaviour at 25", the higher temperature
`favouring greater solubilisation with a small reduction of phosphatide in
`the surface layer. Although this reduction in surface activity appeared
`to continue into the most dilute region examined at 40", there was no
`marked change compared with that observed at 25".
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`SURFACE INTERACTION OF LECITHIN AND LYSOLECITHIN
`Efect of Calcium Chloride
`The lecithin and lysolecithin molecules have two sites of charges at
`the phosphoryl and the cholyl groups. Adsorbed molecules such as soaps
`must also be considered since a primary ageing effect will be hydrolysis at
`the ester linkages.
`Above 0.002 per cent w/v lysolecithin the lowering effect was slightly
`less than in the absence of calcium chloride (Fig. 2). The calcium chloride
`would normally be expected to lower the critical micelle concentration of
`lysolecithin and it was probable that above 0-002 per cent w/v lysolecithin
`the salt assisted aggregation of molecules and consequently increased
`solubilisation of the lecithin. From the small change in surface behaviour
`
`0.01
`
`0.05
`
`34 1
`0
`
`0.002
`
`0.004
`0.006
`0.008
`Concentration of lysolecithin (per cent w/v)
`FIG. 5. Effect of KCI on the surface tension of mixed lysolecithin-lecithin sols at 40".
`For key see Fig. 4.
`it appeared that sufficient lysolecithin was present to prevent substantial
`removal of the lecithin component from the surface layer by calcium
`chloride.
`The instability of the sols over a period of time was believed to be
`brought about by the electrolytes affecting the charge on the colloidal
`particles resulting in a tendency to coagulation.
`On standing for 24 hours the calcium chloride appeared to interact
`with the system in this concentration region of lysolecithin (0.002 per cent)
`resulting in an increase in surface activity of the sols. After another 72
`hours this behaviour spread throughout the concentration range of
`lysolecithin examined. At 40" (Fig. 3) the effect after 24 hours was more
`pronounced but a further 72 hours showed little change. The effect of
`calcium chloride with time on the general behaviour of the system was
`to be expected from the sensitivity of lecithin to this electrolyte. Calcium
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`

`N . ROBINSON
`chloride was active in much smaller concentrations than potassium
`chloride in producing instability of the sols which was thought to be due
`to the divalent calcium ions linking the phosphoryl groups of two single
`molecules or molecules within the aggregates.
`
`E#ect of Potassium Chloride
`The effects of potassium chloride differed from those of calcium chloride
`due largely to the degree of sensitivity of lecithin to these two electrolytes.
`Above a weight fraction of lysolecithin of 0-1 the mixed sols were fairly
`stable to potassium chloride.
`At 25" (Fig. 4) and concentrations greater than 0.0025 per cent wjv lyso-
`lecithin, addition of potassium chloride produced only a slight change in
`surface tension, whereas between 0401 and 0.002 per cent wjv lysolecithin
`the surface tension lowering was considerably increased. This deviation
`was unexpected and thought to arise from ionic forces suppressing the
`aggregation process of lysolecithin molecules. Below this concentration
`region the lowering effect was unchanged. After standing, concentrations
`above 0-002 per cent wjv lysolecithin showed little change but below this
`value the surface tensions steadily increased.
`At 40" (Fig. 5) the effect of potassium chloride was to depress the surface
`activity of the mixed phosphatide system containing more than 0.003 per
`cent w/v lysolecithin but below this concentration there was little change.
`After standing, however, the system showed a considerable lowering in
`the surface tension indicating that removal of lecithin from the surface
`layer took place resulting in small lysolecithin particles producing in-
`creased surface activity.
`The surface activity of the system is thus very sensitive to both calcium
`chloride and potassium chloride particularly in the region of the critical
`micelle concentration of lysolecithin where changes are likely to be
`emphasised. Here the presence of electrolytes shows a tendency to
`increase the surface activity of the mixed phosphatide system, especially at
`40", compatible with changes in metabolism within the environment of a
`cell.
`Acknowledgement.
`
`I thank Dr. Saunders for his interest in this work.
`REFERENCES
`Elworthy and Saunders, J. chem. SOC., 1957, 330.
`Danielli and Davson, J. Cell. Comp. Physiol., 1935, 5, 495.
`Saunders, J. Pharm. Pharmacol., 1957, 9, 830.
`Robinson and Saunders, ibid., 1959, 11, 346.
`Hanahan, Rodbell and Turner, J. biol. Chem., 1954,206,431.
`Robinson and Saunders, J. Pharm. Pharmacol., 1958, 10, 384.
`Lea, Rhodes and Stoll, Biochem. J., 1955, 60, 353.
`Lea Proc. 2nd International Conference on Biochemical Problems of Lipids,
`195s.
`Rhodes, Chem. and Ind., 1956, 75, 1010.
`Perrin and Saunders, J. Pharm. Pharmacol., 1960, 12, 253.
`Neuschloz, Kolloid Z., 1920, 27, 292.
`Price and Lewis, Biochem. J., 1929, 23, 1031.
`Fischgold and Chain, ibid., 1934, 28, 2044.
`Boutaric and Berthier, C.R. Acad. Sci. Paris, 1940, 211, 100.
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