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A new technique for obtaining thin lipid films separating two aqueous media
J. M ol. Biol. (1965) 12, 290-291
A New Technique for obtaining Thin Lipid Films
separating Two Aqueous Media Re centl y a great deal of attention ha s been paid to the formation of thin black films of association colloids between two aqueous phases (Mueller, Rudin, Tien &.Wescott, 1962a,b,1963 ; Mueller & Rudin, 1963; Haydon & Taylor, 1963; Huang , Wh eeldon & Thompson, 1964; H anai, H aydon & Taylor , 1964). For t his work the same t echnique as that originally describ ed by Mueller et at. (1962a ) ha s been used throughout. This technique consists of applying a drop of a solution of the association colloidgenerally lecithin-in hydrocarb on or some other apol ar liquid to a small hole in an insulating wall separating two aqueous media. Drainage and formation of a thin film occur partly by the formation of a torus of the apol ar liquid at the rim of the hole and partly by dissolution of the oil phase in either wall material or aqueous phase. A drawback of this method is that the film so form ed is bound to be small, usually of the order of a few square millimetres. Since films of this kind are of particular interest in the st udy of perm eabilit y, both for electrolytes and non-electrolytes, t his drawback is a serious one. F or small ions the d. c. resist ance has been relied up on as a measure of perm eabili ty (Mueller et al., 1963; Huang et al ., 1964). It t urns out t o be very high . As, mor eover , t he breakdown volt age of films of this kind is fairly low, t he method requires the measurement of sma ll currents, and careful screening is necessar y. Measurement s of the perm eation of non-electrolytes have not been published so far as we know, but in t ha t field too an increase in film size by one or two ord ers of magnitude would be a valu able improvement. An increase in film size of this order can be achieved by passing a hole in a Teflon screen through t he oil- water interphase. The 5-mm t hick screen fits diagonally a 40 mm X 40 mm X 100 mm cuvette. It is essential t hat the hole be carefully machined, giving a sharp edge without irregulari ties. H oles of up to 9 mm diameter hav e been used as well as slits of various widths. Both sides of the film are electrically insulated by using an extra layer of an apolar liquid under the aqueous phase. If the hiller walls of the cuvette ar e made hydrophobic by a resist ant silicone coating, there occurs between the side of the screen and the edge of the cuvett e a capillary rise of the undermost oil and a capillary drop of the upper liquid, both liquids coming easily into contact. Generally the upper oil is a solution of lecithin in heptane or cyclohexane with an admixture of t etradecane to prevent criti cal turbulence during dr ain age. The underlying oil may be chloroform or carbon t etrachloride. To enhance free drainage of the film, the hole is not lowered completely into the water phase but its upper rim is left in t he upp er oil, thus allowing free drainage of t he film into the upper oil phase. Film size may in this way go up to about 50 mm -' without to o much loss of st ability. The whole pro cedure may be changed so that t he lecithin is dissolved in the undermost oil layer and films may be dr awn from there. Using this method we have made numerous measurements, and obtained results similar to those reported by other workers. Drainage is analogous to t he drainage of soap films in air. The boundar y between white and black film is discontinuous. The 290
LETTERS TO THE EDITOR
291
breakdown voltage of a black film lies around 100 mv (Haydon & Taylor, 1963). The film behaves as a purely ohmic resistance. The most accurate resistance measurements are made by determining the current as a function of the applied voltage. In our technique the measuring voltage generally does not exceed 10 mv. The d.c. resistance turns out to be 107 n cm'' for a black film, with some variation between individual films. A sudden fall of resistance, as described by Hanai et al. (1964), is often observed; this could not be correlated with the appearance of black spots or with a sudden increase in the black area. A most remarkable phenomenon is the fall in resistance shortly before the film breaks. Film stability is not markedly affected by large variations in salt concentrations (0 to 0·5 M-KCI, CaCl2 and other salts) or by variations in pH from 2·0 to 10·0. The variation of d.c. resistance with temperature found by T. E. Thompson (communicated at the International Meeting of Biophysics, Paris, 1964) could not be reproduced. Several preparations of phospholipids have been tried; viz. a mixture of ox brain lipids containing lecithin, lysolecithin, cephalin, sphingomyelin and a partially purified mixture of soy bean lipids (asolectin). The most reproducible results were obtained with a preparation of egg lecithin purified by column chromatography (Singleton, Gray, Brown & White, 1964), which was chromatographically homogeneous. Attempts to cover a black film with a polyelectrolyte have so far not been successful. It is known that micelles of an association colloid may interact in several ways with proteins (Green & Fleischer, 1963), the surface area of the association colloid being the main factor governing the detergent/protein ratio of the complex (Booij & Bungenberg de Jong, 1956). Preliminary experiments to study the interaction of a black film with a preparation of structural protein of beef heart mitochondria (Richardson, Hultin & Fleischer, 1964) were seriously hampered by the sensitivity of this kind of film to cholate and deoxycholate, which are necessary to solubilize the protein. H. J.
VAN DEN BERG
Philips Research Laboratories N.V. Philips' Gloeilampenfabrieken Eindhoven-Netherlands Received 28 January 1965 REFERENCES Booij, H. L. & Bungenberg de .Iong, H. G. (1956). Biocolloids and their Interactions, pp. 112-147. Vienna: Springer Verlag. Green, D. E. & Fleischer, S. (1963). Biochirn. biophys. Acta, 70, 554. Hanai, T., Haydon, D. A. & Taylor, J. (1964). Proc, Roy. Soc. A, 281, 377. Haydon, D. A. & Taylor, J. (1963). J. Theoret. Biol. 4, 281. Huang, L., Wheeldon, L. & Thompson, T. E. (1964). J. Mol. Biol. 8, 148. Mueller, P. & Rudin, D. O. (1963). J. Theoret. Biol. 4,268. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1962a). Nature, 1964, 979. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1962b). Oirculation, 26, 1167. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1963). J. Phys. Ohern. 67, 534. Richardson, S. H., Hultin, H. O. & Fleischer, S. (1964). Arch. Biochem, Biophys. 105, 288,454. Singleton, W. S., Gray, M. S., Brown, M. L. & White, J. L. (1964). J. Amer. oa ou-: Soc. 41, 16. 19*
A New Technique for obtaining Thin Lipid Films
separating Two Aqueous Media Re centl y a great deal of attention ha s been paid to the formation of thin black films of association colloids between two aqueous phases (Mueller, Rudin, Tien &.Wescott, 1962a,b,1963 ; Mueller & Rudin, 1963; Haydon & Taylor, 1963; Huang , Wh eeldon & Thompson, 1964; H anai, H aydon & Taylor , 1964). For t his work the same t echnique as that originally describ ed by Mueller et at. (1962a ) ha s been used throughout. This technique consists of applying a drop of a solution of the association colloidgenerally lecithin-in hydrocarb on or some other apol ar liquid to a small hole in an insulating wall separating two aqueous media. Drainage and formation of a thin film occur partly by the formation of a torus of the apol ar liquid at the rim of the hole and partly by dissolution of the oil phase in either wall material or aqueous phase. A drawback of this method is that the film so form ed is bound to be small, usually of the order of a few square millimetres. Since films of this kind are of particular interest in the st udy of perm eabilit y, both for electrolytes and non-electrolytes, t his drawback is a serious one. F or small ions the d. c. resist ance has been relied up on as a measure of perm eabili ty (Mueller et al., 1963; Huang et al ., 1964). It t urns out t o be very high . As, mor eover , t he breakdown volt age of films of this kind is fairly low, t he method requires the measurement of sma ll currents, and careful screening is necessar y. Measurement s of the perm eation of non-electrolytes have not been published so far as we know, but in t ha t field too an increase in film size by one or two ord ers of magnitude would be a valu able improvement. An increase in film size of this order can be achieved by passing a hole in a Teflon screen through t he oil- water interphase. The 5-mm t hick screen fits diagonally a 40 mm X 40 mm X 100 mm cuvette. It is essential t hat the hole be carefully machined, giving a sharp edge without irregulari ties. H oles of up to 9 mm diameter hav e been used as well as slits of various widths. Both sides of the film are electrically insulated by using an extra layer of an apolar liquid under the aqueous phase. If the hiller walls of the cuvette ar e made hydrophobic by a resist ant silicone coating, there occurs between the side of the screen and the edge of the cuvett e a capillary rise of the undermost oil and a capillary drop of the upper liquid, both liquids coming easily into contact. Generally the upper oil is a solution of lecithin in heptane or cyclohexane with an admixture of t etradecane to prevent criti cal turbulence during dr ain age. The underlying oil may be chloroform or carbon t etrachloride. To enhance free drainage of the film, the hole is not lowered completely into the water phase but its upper rim is left in t he upp er oil, thus allowing free drainage of t he film into the upper oil phase. Film size may in this way go up to about 50 mm -' without to o much loss of st ability. The whole pro cedure may be changed so that t he lecithin is dissolved in the undermost oil layer and films may be dr awn from there. Using this method we have made numerous measurements, and obtained results similar to those reported by other workers. Drainage is analogous to t he drainage of soap films in air. The boundar y between white and black film is discontinuous. The 290
LETTERS TO THE EDITOR
291
breakdown voltage of a black film lies around 100 mv (Haydon & Taylor, 1963). The film behaves as a purely ohmic resistance. The most accurate resistance measurements are made by determining the current as a function of the applied voltage. In our technique the measuring voltage generally does not exceed 10 mv. The d.c. resistance turns out to be 107 n cm'' for a black film, with some variation between individual films. A sudden fall of resistance, as described by Hanai et al. (1964), is often observed; this could not be correlated with the appearance of black spots or with a sudden increase in the black area. A most remarkable phenomenon is the fall in resistance shortly before the film breaks. Film stability is not markedly affected by large variations in salt concentrations (0 to 0·5 M-KCI, CaCl2 and other salts) or by variations in pH from 2·0 to 10·0. The variation of d.c. resistance with temperature found by T. E. Thompson (communicated at the International Meeting of Biophysics, Paris, 1964) could not be reproduced. Several preparations of phospholipids have been tried; viz. a mixture of ox brain lipids containing lecithin, lysolecithin, cephalin, sphingomyelin and a partially purified mixture of soy bean lipids (asolectin). The most reproducible results were obtained with a preparation of egg lecithin purified by column chromatography (Singleton, Gray, Brown & White, 1964), which was chromatographically homogeneous. Attempts to cover a black film with a polyelectrolyte have so far not been successful. It is known that micelles of an association colloid may interact in several ways with proteins (Green & Fleischer, 1963), the surface area of the association colloid being the main factor governing the detergent/protein ratio of the complex (Booij & Bungenberg de Jong, 1956). Preliminary experiments to study the interaction of a black film with a preparation of structural protein of beef heart mitochondria (Richardson, Hultin & Fleischer, 1964) were seriously hampered by the sensitivity of this kind of film to cholate and deoxycholate, which are necessary to solubilize the protein. H. J.
VAN DEN BERG
Philips Research Laboratories N.V. Philips' Gloeilampenfabrieken Eindhoven-Netherlands Received 28 January 1965 REFERENCES Booij, H. L. & Bungenberg de .Iong, H. G. (1956). Biocolloids and their Interactions, pp. 112-147. Vienna: Springer Verlag. Green, D. E. & Fleischer, S. (1963). Biochirn. biophys. Acta, 70, 554. Hanai, T., Haydon, D. A. & Taylor, J. (1964). Proc, Roy. Soc. A, 281, 377. Haydon, D. A. & Taylor, J. (1963). J. Theoret. Biol. 4, 281. Huang, L., Wheeldon, L. & Thompson, T. E. (1964). J. Mol. Biol. 8, 148. Mueller, P. & Rudin, D. O. (1963). J. Theoret. Biol. 4,268. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1962a). Nature, 1964, 979. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1962b). Oirculation, 26, 1167. Mueller, P., Rudin, D.O., Tien, H. Ti & Wescott, W. C. (1963). J. Phys. Ohern. 67, 534. Richardson, S. H., Hultin, H. O. & Fleischer, S. (1964). Arch. Biochem, Biophys. 105, 288,454. Singleton, W. S., Gray, M. S., Brown, M. L. & White, J. L. (1964). J. Amer. oa ou-: Soc. 41, 16. 19*