PERMEABILITY OF MEMBRANES1 BY WILDER D. BANCROFT AND CHARLES GURCHOT
We call a membrane semipermeable when it lets one constituent of a solution pass through-usually water- and does not permit diffusion of the other constituent or of one or more of the other constituents. It has usually been assumed tacitly that the membrane remained semipermeable unless it was ruptured mechanically. There is already a good deal of evidence to show that this simple view is not adequate. Bariow2 has shown that alcohol makes a copper ferrocyanide membrane permeable to sugar though he attempts no explanation of the phenomenon. I n fact he apparently did not test for the sugar but merely noted the decrease of osmotic pressure when the cell containing a sugar solution was placed in aqueous alcohol. “Experiments were performed with varying strengths of solutions, going up from fifteen percent alcohol by steps of five percent to seventy percent. Strengths below and above these limits were also used, all with the same result. This gradual increase in the strength of the solution was thought to be advisable, because it seemed not unlikely that when the alcohol was in great excess the sign of the osmotic current might change. ID other words, the alcohol might become the solvent and the water the solute. This, however, was not found even when the water was present in very small quantity indeed.” Since sugar is insoluble in alcohol, it is clear that the alcohol cannot make the copper ferrocyanide membrane permeable to sugar by increasing the solubility of the sugar in the membrane. There must therefore have been a fundamental change in the membrane. Another unexplained phenomenon is one studied by Czapek3, a number of years ago. He found that there was an exosmosis with an accompanying transfer of tannin from cells of Echeveria when these were placed in aqueous solutions of different alcohols. The crjtical concentrations a t which exosmosis took place varied at 15’-19’ from fifteen volume percent with methyl alcohol to 10-11 percent with ethyl alcohol, 4-5 percent with isopropyl and normal propyl alcohols, 1-2 percent with isobutyl and normal butyl alcohols, and 0.5 percent with amyl alcohol. These are, however, the concentrations at which the surface tension of water has been lowered to 68-69 percent of its normal value. It is therefore sometimes stated that permeability occurs when the surface tension of the water has been reduced to about two-thirds of its normal value. This is going beyond the facts as now known, because allyl alcohol and tertiary butyl alcohol become effective a t different surface tensions. 1 Preliminary paper presented before Section B of the British Association for the Advancement of Science a t the Toronto meeting. PPhil. Mag. (6) 10, I ; 11, 595 ( 1 9 ~ 6 ) . ‘Ber. deutsch. bot. Ges. 28, 159 (1910).
1280
WILDER D. BANCROFT AND CHARLES GURCHOT
Czapek considers that the behavior of these last two alcohols may be due t o some secondary toxic action. “A good example of such secondary toxic actions occurs with acetic acid which causes permeability before the critical concentration [with reference to surface tension] has been reached because of the toxic action due to its being an acid. For the theory of narcotics it is especially important to establish that a large number of these substances become toxic when a surface tension is reached characteristic for each organism. Among these substances are to be classed the saturated alcohols with normal carbon chain; and also acetone and ethyl acetate. Among the esters there are a number, notably methyl acetate and ethyl formate which are characterized by special toxic action. “In the typical alcohol action we are certainly dealing chiefly with a diffusion phenomenon. The tannin can only begin to diffuse outward when its solubility in the plasma film and in the external medium is somewhat. greater than in the cell solution’. If this becoming soluble coincides with changes in the surface tension of all three media (outer liquid, plasma film, and liquid inside the cell), one seems justified in aesuming that a t the critical concentration [at which exosmosis begins] the surface teneions of the plasma film and the outside solution have become equal. “If the pbsma film were a chemically and physically homogeneous substance, our method would give us a means of determining the surface tension of the plasma film. It is not possible, however, to simplify to such an extent the factors governing diffusion in the cell. From what we know, the plasma film is a complicated, heterogeneous, colloidal film, in which, as Overton has shown so convincingly, lipoid-soluble substances play an important part. The increased permeability of the plasma film to the substances in the cell solution with lowered surface tension of the outer liquid may well be due to changes in the plasma film colloids a t the surfaces in question. Perhaps somebody will later formulate this problem more exactly.” If Csapek had known that tannin is only in colloidal solution, he would probably have been able to take the next step himself. It does not seem t o have occurred to him, however, to make any experiments to determine whether there had or had not been any permanent change in the membrane. Walden2 showed that tenth-normal solutions of formic, acetjc, propionic, butyric, isobutyric, valerianic, cyanacetic, halogen-substituted acetic, glycollic, glyoxalic, methyglycollic, ethylglycollic, glyceric, alpha-oxybutyric, beta-oxybutyric, meta-oxybenzoic, para-oxybensoic, diglycollic, quinic, tartaric, citric, mandelic, alpha-nitrophthalic, mellitic, tetra-carbonic, dimethylmalonic, quinolinic, acrylic, and caproic acids passed through the copper ferrocyanide membrane while the sodium salts of most of these acids did not. This is a very strange thing, because there is no apparent reason why organic acids should be soluble in copper ferrocyanide. As Walden was working empirically with no theory to guide him, it did not occur to him to test whether 1
[There seems to be no justification for this statement.] 10, 705 (1892).
Z. physik. Chem.
PERMEABILITY O F MEMBRANES
1281
the organic acids had, by any chance, caused any permanent change in the membrane. Czapekl found that his plant cells (Echeveria) gave the exosmose reaction with concentrations of hydrochloric, sulphuric, nitric, phosphoric, oxalic, acetic, malic, tartaric, citric, lactic, fumaric, and salicylic acids at concentration? exceeding N/h,too. It seems very improbable that a reaction involving hydrogen ion should occur a t equimolecular concentrations of these acids and there is either a large experimental error or else some factor has been overlooked. When we consider the permeability of the living cell, we find that no simple theory of permeability is adequate and that it is apparently necessary to postulate that a given cell* membrane is both permeable and impermeable for the same substance. A lipoid film will account for a great many of the phenomena; but it would not let water through, so we have to postulate an action due to lecithin. A great many of the acid dyes are insoluble in lipoids but penetrate certain cells readily. Worse than that, one finds that among the substances for which the cells are normally impermeable are grape sugar, fruit sugar, cane sugar and other carbohydrates, the amino-acids and the acid amides, and many other substances which are foodstuffs for the cells and which are brought into the cells from the outside. Hober says, rather despairingly, that “what the cell can use, it shuts out, and what it cannot use, i t lets in.” This difficulty disappears completely if we assume that the permeability of the cell membrane depends on the surrounding medium and that relatively slight changes in the latter may make the cell membrane permeable or impermeable to a given substance. There is nothing especially new about this hypothesis. Years ago, Hober3 suggested that the cell might be impermeable while a t rest and permeable when active. The same point of view is apparently held by R. S. Lillie4 and by Baylisss. “It is a matter of great difficulty to suggest any probable structure for the membrane, Owing to its mode of production, it is no doubt of a very complex chemical nature. It appears to be in all states of the cell permeable to all substances soluble both in water and in oil or organic liquid, such as urea, some ammonium salts, alcohol, chloroform, carbon dioxide, oxygen, etc., as Meyer and Overton pointed out. I n the resting state of the cell, the membrane is impermeable to salts, other than certain salts of ammonium, to glucose and to aminoacids, while in a state of activity it becomes permeable to all these.” The difficulty with this is that it is vague6 and that there is no independent proof of the hypothesis as yet. If we consider a copper ferrocyanide membrane as a colloidal film-which is what it is-it is evident that anything Ber. deutsch. bot. Ges. 28, 161 (1910). Hober: “Physikalische Chemie der Zelle und Gewebe,” 476-544 (1922). “Physikalische Chemie der Zelle und Gewebe,” 263 (1911). “Protoplasmic Action and Nervous Action,” 347, 356 (1923). “Interfacial Forces and Phenomena. in Physiology,” 134 (1922). Lillie: “Protoplasmic Action and Nervous Action,” 337 (1923).
I282
WILDER D. BANCROFT AND CHARLES GURCHOT
which will coagulate the film will make it permeable. It seemed probable therefore that Barlow’s experiments with sugar and alcohol could be explained ori the assumption that alcohol coagulated the membrane. This was a point which could easily be tested experimentally. Copper ferrocyanide membranes were made by a modification of Collander’s method. Glass tubes were used, 3 cm in diameter and I O cm long. One end was ground on each tube and covered with a layer of cheesecloth to serve as a support for the membrane. This end was dipped into a warm, ten percent, solution of gelatine freed from air bubbles by filtering through sand. The gelatine was allowed to dry on the cheesecloth, the tube being held downward after removing the excess of gelatine. It is immaterial if a few air bubbles persist in the gelatine film a t this stage. When the gelatine film was dry, 2cc of the same gelatine solution, freed from air bubbles: were introduced inside the tube on top of the first gelatine layer. It is important that there are no bubbles in this layer. This upper layer was allowed to set to a stiff jelly but not to become bone dry and was hardened for twenty-four hours in a two percent solution of formeldehyde. The gelatine film wae washed free of formaldehyde in several changes of distilled water, the tubes being allowed to stand for about two hours in each change of water. The tubes were then suspended in tumblers or beakers by means of large corks bored to fit the glass tubes. A fresh solution containing 0.02 mols potassium ferrocyanide was placed outside the membrane and a solution containing 0 . 0 2 mols copper sulphate was put inside the tube. I n about half an hour, copper ferrocyanide began to form on the inner surface as a uniform, brown membrane. The reaction was allowed to run for twenty-four hours. Each membrane was tested by putting a one percent solution of cane sugar inside the tube and distilled water outside. The membrane was considered satisfactory if there was no test for sugar in the outside water after sixteen hours. When a sugar solution was put on one side of the membrane and a dilute methyl alcohol solution on the other side, sugar passed through the membrane, thus confirming Barlow’s results, Since the question of permeability may be to some extent dependent on the time factor, tests for the presence or absence of sugar were always made at the end of about sixteen hours. TO show that a permanent change in the membrane had taken place, the membrane was exposed to aqueous methyl alcohol solutions and the membrane was washed with water. It was then found to be permeable to sugar in the absence of alcohol, showing that coagulation had taken place. Less than two percent of methyl alcohol are sufficient to make the copper ferrocyanide membrane permeable to sugar. With the higher alcohols the necessary concentrations were much lower; but we are not prepared as yet to say t o what extent the critical concentrations correspond to equal surface tensions. The results are in qualitative agreement, however, with those of Caapek and we feel certain that the permeability which he obtained with Echeveria was due to coagulation of the cell membrane. A corollary from this is that methyl alcohol must coagulate a copper ferrocyanide sol and this proved to be true. The precipitation of the sol
PERMEABILITY OF MEMBRANES
1283
is a much less sensitive test than the one for the permeability of the membrane and it takes approximately forty percent methyl alcohol to coagulate a copper ferrocyanide sol under the conditions which we employed. Experimental details will be given in a later paper by Mr. Gurchot. Similar experiments were also made with copper ferrocyanide membranes and solutions of acetic acid so as to confirm Walden’s results. When solutions of acetic acid stronger than 0.5 percent have been in contact with a copper ferrocyanide membrane, the membrane i s no longer impermeable to sugar and has therefore coagulated. In line with this is the fact that acide precipitate a copper ferrocyanide sol and alkalies peptize it. It is possible that the permeability of cell membranes after death is due to the increased acidity of the system. A few experimentg were made to regenerate the membrane by changing from an acid solution to an alkaline one; but no eatisfactory results have been obtained. This is not essential because the materials which form the membrane in a plant cell or an animal cell are probably being supplied continually. A more interesting line of attack will be to repeat the preceding experiments with a little copper salt on one side of the membrane and a little ferrocyanide on the other side. Experiments of this sort will be started a t once. The general results of this preliminary paper are as follows: I . Copper ferrocyanide membranes are coagulated by low concentrations of methyl alcohol and of acetic acid, thereby becoming permeable to sugar. 2 . Barlow’s experiments with alcohol, Walden’s experiments with acids, and Czapek’s experiments with alcohols and acids involved nothing more mysterious than a coagulation of the membrane. 3. Alcohols and acids coagulate a copper ferrocyanide sol while alkalies peptize a copper ferrocyanide gel. Cornell Uniitersitu.
