REVERSIBLE PERMEABILITY OF hlEWIBRAXES AiYD rrrs RELATION TO CELL METABOLISM BY CHARLES GURCHOT
Introductory
It is well-known that a living cell removed from its parent organism can be kept alive in the proper liquid medium. If, instead of a single cell, a community of cells or a piece of tissue is kept in a nutritive liquid, the tissue not only will be preserved but it will grow and proliferate. In other words, in its own small way, it behaves more or less as a complete organism as long as the medium remains constant and as long as the tissue remains the same as it was a t the start. But the isolated cell behaves differently. Its isolation is tantamount to a physiological divorce in addition to an anatomical one for the cell appears to be permanently a t rest and in this condition it is impermeable for almost everything which it ordinarily uses for food and t o the products of its metabolism which are formed in the interior of the cell1 “from the undissolved carbohydrate reserves, and also for inorganic salts and salts of organic acids”. Naturally the cell cannot behave in this way when still a part of the original organism. Another significant phenomenon takes place after death. Then the cell behaves as an ordinary membrane which retains colloids only. It seems fairly well established, now, that the cell is surrounded by a pellicle which functions as a semi-permeable membrane. Deductions from the second law of thermodynamics embodied in Willard Gibbs’ principle show that all substances present in the cell and which lower its surface tension must concentrate at the periphery, that js at the interface between the cell contents and the surrounding medium2. It has been shown that the concentration of some bile salts may exceed the solubility3. Such substances will be deposited then as solid films enclosing the cell. One is forced to ask whether something does not take place in the mass of tissue which alters the permeability of the cell through something which is either stored or produced in cells en masse. Unfortunately it is impossible at the present time to exact an equivocal answer to this question from the cell directly, for the simple reason that the composition of cell membranes is not known. Yet previous considerations suggest that we ought to find in the membrane substances which lower the surface tension of water considerably. Such are the fatty compounds. Nor is it known what makes the membrane semi-permeable, let alone how the permeability is altered. It is necessary to work backward, so to speak, and to proceed by analogy. It seems advisable therefore to consider the whole question of semi-permeable membranes and perhaps of membranes in general. Experiments must be Bechhold: “Colloids in Biology and Medicine”, 214 (1919). Bayliss: “Principles of General Physiology” (1918). Ramsden: Proc. Roy. Soc., 72, 156 (1904).
84
CHARLES GURCHOT
made first on easily reproducible membranes of definite composition and for which the technique has been worked out already. Copper ferrocyanide offers a membrane of definite composition which in many ways behaves not unlike living semi-permeable membranes. Copper ferrocyanide has been the subject of painstaking investigations of which those of Walden’ are among the most important. Walden investigated the action of many inorganic and organic salts and acids on several membranes without, however, observing to see whether any of the substances which passed through had a permanent effect. It is true that his method of procedure made such observation impossible. Walden always had the reagents with which he formed his membranes on each side of them. I n this way the membranes were constantly regenerated and the observation of permanent injury was out of the question. This same mode of manipulation enabled Collander,2 recently, to reach unwarranted conclusions regarding the behavior of living membranes. We are confident that, in order to pursue its metabolic functions, the cell must, in some way, change its permeability; and, in fact, we know that it does. The larval cells of Arenicola when in a state of contraction lose the pigment ordinarily present in the cells’. Pilocarpine makes certain cells more permeable, atropine makes them less permeable, to certain dyes4. The egg cell on fertilization and the muscle cell on contraction increase their electrical conductivity5. More than this we have evidence that permeability changes are reversible. One example will suffice. The seaweed Laminaria increases its electrical conductivity when immersed in a ca. 2 . 5 7 0 solution of pure sodium chloride. On adding a little calcium chloride to the solution Laminaria returns to normal conductivity without showing signs of permanent damage6. Clowes’ offers an explanation which, although insufficient, is nevertheless an advance over previous ones. He compares the cell membrane to an oil-water emulsion in which soaps concentrate at the interface between oil and water and lower the surface tension of one or the other liquid depending on whether or not there is present an oil or a water-soluble soap. Clowes goes on to summarize Bancroft’s explanation for emulsions as follows :-“soaps of monovalent cations being readily dispersed in water but not in oil form a film or diaphragm which is wetted more readily by water than by oil, consequently the surface tension is lower on the water than on the oil side. Since the area of the inside surface of a film surrounding a sphere is smaller than that of the outside surface, the film tends to curve so that it encloses globules of oil in water, in this manner reducing the area of the side of greater surface tension to a minimum as compared with that of lower surface tension. On the other hand B film composed of soaps of divalent or trivalent cations being freely disZ. physik. Chem. 10, 699 (1892). Kolloidchem. Beihefte, 20, 273 (1925). 3 Lillie: Am. J. Physiol. 37, 764 (1913). 4 Garmus: Z. Biol. 58, 185 (1912). 5 McClendon: Am. J. Physiol., 27, 240 (1910); 29, 302 (1912). Science, ( 2 ) 34, 187 (1911). 7 J. Phys. Chem., 20, 407 (1916). 2
REVERSIBLE PERMEABILITY O F MEMBRANES
85
persed in oil but not in water is wetted more readily by the oil than by the water, the surface tension is lower on the oil than on the water side, and the film tends to curve in such a manner as to enclose the globules of water in an outer or continuous oil phase”. The process1 “is dependent on the presence of a film of soap between the phases and it is interesting to note the powerful effect of sodium oleate in destroying red blood corpuscles”. On the addition of calcium chloride the resulting water in oil emulsion “would be impermeable to water but permeable to substances soluble in oil. A change of the latter kind if complete would not give us the properties which the normal cell-membrane possesses”. It would be permeable to water and its solutes only on reversal to the oil in water emulsion. Such a membrane would be entirely too labile to enable the cell to manifest the osmotic pressure which it has actually. It is perhaps better to conceive of the cell membrane as a more or less rigid colloidal suspension, irreversible as we understand an emulsion to be reversible, but, as we shall see later, possessing a reversihility of its own. We shall see also that the degree of permeability can be altered in such a membrane. Another interesting feature of the work of Clowes is the analogy which is drawn between the elongated drop stage, in the course of the reversal of the emulsion, when the dispersing liquid flows as through narrow channels, and the porous sieve-like character of the copper ferrocyanide membrane as conceived by TraubeY. A great deal has been done to prove that water flows through a copper ferrocyanide membrane as it would through a series of capillary tube^.^ Poiseuille’s equation4 has been called upon to prove this: Q = - KD4PT L where Q is the amount of liquid, K a constant for a definite temperature, D the diameter of the pores, P the pressure applied, and L the length of the capillaries. From this equation values have been calculated for the diameter of the pores. It is unfortunate that Poiseuille’s equation should have been misused in this way. The equation expresses exactly the results of motion of a viscous fluid through pipes of circular cross-section. It is assumed that the velocity is everywhere perpendicular to the cross-section of the pipe and it has been pointed out by Lamb that if any appreciable amount of slipping a t the boundary of the pipes, used by Poiseuille, took place a deviation from the law of the fourth power of the diameter would become apparent. It becomes perfectly clear, therefore, that the differencein pressure between the inlet and the outlet of the pipe or tube is not an accurate measure of the pressure gradient because some of this pressure difference is required to give kinetic energy of niotion to the water in the eddy currents which will be formed whenever the diameter of the pipe or tube is not uniform throughout. It is more than probable that this state of affairs obtains in the case of semi-permeable membranes. Bayliss: “Third Report on Colloid Chemistry”, 4 (1921). Archiv fur Anatomie, 1867, 67. 3Bartell: J. Phys. Chem. 15, 659 (1911); 16, 318 (1912). Poiseuille: Compt. rend. 11, (1840); 12, (1841). 1
86
CHARLES GURCHOT
I n other words if the equation for Bartell’s membranes containcd the term D raised to some other power than the fourth his results would have been the same. But as semi-permeable membranes generally operate a t approximately atmospheric pressure and especially in the presence of electrolytes it might have been better to employ Fick’s diffusion equation: dS - _ - k q-dC where dS/dt is the rate of flow, k the temperature codt dX efficient, q the cross-section of the pores, dC/dX the rate of change in concentration of the diffusing solution. But after all has been said there is no reason to suppose that a semi-permeable membrane does not behave as if it were porous. After all, inter-molecular spaces are only a special type of pores. But we know that other factors, such as selective adsorption, affect the permeability of a membrane. It follows from this that both Poiseuille’s and Fick’s equations are useless to express the true diameter of pores even if it is granted that we have to deal with uniform capillaries. This we know is not true. Consequently all we can say is that copper ferrocyanide is porous. We know that this is vague to say the least. We also know that the statement does not mean anything for, strictly speaking, the water membrane between chloroform and ether is porous also. Attempts have been made to prove the copper ferrocyanide membrane to be an atomic sieve from the fact that progressively larger molecules diffused with increasing difficulty. Such a simple view is entirely inadequate a t the present time. There is evidence to show that a copper ferrocyanide membrane does not work as a static system but rather as a dynamic one. When Czapek’ exposed cells of Echeveria to the action of various alcohols he noticed that an exosmosis took place of the tannin present in the cells. For methyl alcohol the critical concentration a t 15-19OC. was about 15% by volume, for ethyl I O - I I % , propyl 4-5yc,butyl 1-20/~,amyl 0.5%. Because the above solutions possessed a surface tension of about 68% that for pure water Czapek concluded that the surface tension lowering WAS responsible for the exosmosis, 68% of the value for water being the critical tension. This was as far as Cznpek ventured. Had he known that tannin exists in colloidal solution he might have guessed that the exosmosis was due to coagulation of the cell membrane. Czapek found also thal his plant cells gave the exosmose reaction with various organic and inorganic acids for concentrations exceeding ?T/6400. This was undoubtedly another case of coagulation owing to the adsorption of hydrogen ions by the cell membrane. In 1906 Barlow2,in the course of a series of osmotic pressure measurements through copper ferrocyanide, observed that the addition of alcohol to the outside solution caused a lowering of the osmotic pressure. Fearing that with alcohol in great excess the sign of the osmotic current might change, Barlow looked upon this phenomenon as an unfortunate incidenb-the alcohol having spoiled his membrane. Ber. deutsch. botan. Gaz. 28, I j 9 (1910). Mag. 16) 10, I ; 11, 595 (1906).
* Phll.
REVERSIBLE PERMEABILITY O F MEMBRANES
87
The whole question of membrane action seems to involve selective adsorption and coagulation; and the natural corollary is that the coagulation is reversible. There is no question that an investigation in this direction will throw some light a t least on the behavior of cell membranes. In fact it seems very probable that our views on membrane action in general must undergo considerable revision. The experiments described in the following pages will show that a copper ferrocyanide membrane behaves as a colloidal film analogous to a colloidal sol. It can be coagulated andit can also be peptized. @nthe other hand the peculiar spatial relations of a film in contrast to a sol make the consequences arising from the analogous behavior of the former very significant when we consider the reactions of living membranes. Such a film possesses, for example, a certain amount of rigidity and inertia which, to be sure, are essential if in the cell the film is to play the part of metabolic censor, so to speak. But as we shall see later, such properties make it very difficult to prove the analogy by experiment. If a continuous film-it is better to say a film with very fine interstices-can be converted at will into a sieve whose meshes can be closed when necessary we have here an answer to the question why a substance which ordinarily does not pass through a membrane will do so, nevertheless, a t certain times. The antagonistic action of a peptizing agent toward a coagulating agent will enable us to explain the sodium-calcium antagonism in living cells. Of course it must not be expected that sodium and calcium will be antagonistic for copper ferrocyanide. There is every reason why they should not be. The antagonism will be of a different kind. After all, whatever may be the composition of cell membranes we are quite certain it is not copper ferrocyanide.
Experimental Copper ferrocyanide membranes were made by a modification of Collander’s method. Glass tubes were used, 3cm. in diameter and Iocm. long. One end of each tube mas ground and covered with a layer of cheese cloth to serve as a support for the membrane. This end was dipped into a warm ten per cent solution of gelatine freed from air bubbles by filtering through sand. The gelatine solu,tion should not be heated higher than 4oOC. Instead of a ten per cent a fifteen percent solution of gelatine may be used. It will give a firmer support and will be very much less liable to damage; but a t the same time the membrane will take longer to be deposited in the denser gelatine layer where diffusion must take place more slowly. The gelatine was allowed to set to a stiff jelly on the cheese cloth, the tube being held downward after the excess of gelatine had been removed. It was found convenient to hasten the jellying by blowing compressed air on the tube for about a half minute. It is immaterial if a few air bubbles persist in the gelatine film at this stage. When the gelatine film was a firm jelly, 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. The reason is, of course, that a bubble in the plane of the deposited membrane will destroy its continu-
88
CHARLES GURCHOT
ity. This upper layer was allowed to set to a stiff jelly also and it was hardened for twenty-four hours in a two percent solution of formaldehyde, The gelatine tubes were washed free of formaldehyde in several changes of distilled water, the tubes being allowed to remain about two hours in each change of water. They were suspended in tumblers or in beakers by means of two copper strips, about one centimeter wide, pinched at each end with paper clips. A small piece of Gooch rubber tubing at the top of each tube prevented the latter from slipping through the copper strips whose fastened ends rested on the edge of the tumbler or the beaker. A fresh solution containing 0 . 0 2 mols potassium ferrocranide was placed out,side and a solution containing 0.02 mols copper sulphate was put inside the tube. In about a half hour copper ferrocyanide began to form near the inner gelatine surface as a uniform brown membrane. The reaction was allowed to go on for twenty-four hours if a ten percent gelatine solution was used. The reaction required forty-eight hours in case a fifteen percent gelatine solution was employed. After each membrane had been washed free of the reagents it was tested by putting a one percent solut i m of cane sugar inside the tube and distilled water outside. If there was no test for sugar in the outside water after sixteen hours, the membrane was considered satisfactory. The above procedure was carried out at room temperature, that is to say about 2oOC. As a result never more than about two-thirds of the membranes made were found satisfactory. But as we shall see later certain considerations necessitated a change in method, and when the membranes were formed and kept at 8°C. the yield was one hundred percent and they lasted much longer. It is sometimes advised that thymol be added to the gelatine solution to prevent the membrane from being destroyed in the first process by bacteria which liquefy gelatine. This added inconvenience is unnecessary provided cleanliness is observed. There are bacteria which attack the copper ferrocyanide film and create holes in it. But by working a t 8°C. this contingency is prevented also.
Coagulation with Propyl Alcohol The first thing to be investigated was the permanent action of alcohols on the copper ferrocyanide film, For this, twelve series of two tubes were used. I n every test it must be assumed that three membranes were used unless it is stated otherwise. Propyl alcohol of varied strengths was put in each tube. Distilled water was put outside. At the end of sixteen hours, after the membranes had been washed thoroughly, a one percent solution of sugar was introduced inside each tube. The water outside was tested for the presence of sugar sixteen hours later. The semi-quantitative results are given in Table I. It is interesting to note that greater strengths of alcohol were not followed with a more abundant Fehling precipitate. This is just what should be expected if the copper ferrocyanide film is conceived to be a close aggregation of colloidal particles each surrounded with a film of adsorbed water. On addition of alcohol the individual particles-or the small groups-unite to form larger aggregates, This is followed by an increase in porosity sufficient to let
REVERSIBLE PERMEABILITY O F MEMBRANES
89
TABLE I Membranes
Propyl alcohol % by volume
I
I
2
1.5
3 4
2
5 6 7 8
3.0
9
.o
2 . 5
3.5 4.0 4.5 5.0
IO
5.5
I1
6.0
Precipitatc with Fehling’s solution
Faint trace Large trace Moderate Moderate Large trace Large trace Moderate Moderate Trace Trace Trace
sugar through. Rut it must be much more difficult for these aggregates to unite and form still larger ones. To accomplish this it may be necessary to use much higher concentrations of alcohol. It is even possible that .the resistance of the gelatine will prevent further coagulation even then. If the coagulation of the membrane is an effect of the decrease in surface tension brought about by adding alcohol to water it is conceivable that coagulation might be reversed by bringing the membrane in contact with a liquid of higher surface tension, namely pure water. Accordingly the alcohol-treated membranes were exposed to water about forty-eight hours. At the end of that time no reversibility had taken place. The membranes were still permeable to sugar. A second treatment with water gave no better results. This, of course, does not prove that surface t,ension is not a factor in the coagulation of copper ferrocyanide.
Coagulation with Methyl Alcohol The experiments were repeated, this time methyl alcohol was used because it is available for smaller changes of surface tension than propyl alcohol. The alcohol and the sugar were put inside the tube a t the same time. The results are given in Table 11. TABLE I1 Alcohol inside tube; sugar solution IY&exposure 16 hours; room temperature 2oOC. Membrane yo Methyl alcohol Fehling’s precipitate I 0. I n’one 2 0.2 None None 3 0.3 None 4 0.4 5 0.6 None 6 0.8 None 7 1.0 None 8 1.5 Faint trace The minimal concentration for methyl alcohol seems to be about, 1.5%
90
the
CHARLES GURCHOT
The experiments were repeated with methyl alcohol outside the tJubeand 1% sugar solution inside the tube. The results are given in Table 111.
TABLE I11 Alcohol outside tube; sugar solution 1% room temperature 2oOC. Membrane % Methyl alcohol I
0.I
2
0.5
3 4
I
5
2.0
.o
1.5
inside tube; exposure 16 hours; Fehling’s precipitate
None None None None Trace
The noteworthy difference is that more alcohol was necessary to coagulate the membrane here than when both sugar and alcohol were on the same side, This is not surprising. Since sugar is insoluble in pure alcohol the addition of the former to an aqueous alcoholic solution must raise the partial pressure of the alcohol. In other words when sugar and alcohol are on the same side of the tube alcohol behaves as if it were more concentrated. Both series of membranes were also given the water treatments of fortyeight hours each, but no recovery took place. If, as was euggested, coagulation is a surface tension effect-which is doubtful as we shall see laterreversibility must nevertheless be difficulc to obtain inside the gelatine support. Here something must be said about the question of minimal concentration. I t is not devoid of ambiguity because just as soon as a membrane becomes ever so slightly permeable to sugar the permeability will not be apparent until the membrane has been exposed to sugar for a sufficient length of time. In addition to this a very long exposure to very dilute alcohol will show the same results as a correspondingly short exposure to more concentrated alcohol, The time must therefore be fixed arbitrarily, and the minimal concentration must be allowed to remain a function of the time, the temperature remaining constant. It is practically impossible, consequently, to know just exactly when a membrane becomes permeable to sugar. And in addition to this it is not a t all surprising that reversibility, if it can be obtained a t all, by increasing surface tension, is difficult to accomplish. Coagulation and Reversibility of Copper Ferrocyanide Sol
A corollary from the above experiments is that methyl alcohol must coagulate a copper ferrocyanide sol. But the precipitation of the sol proved to be much less sensitive than the test for the permeability of the membrane. In the case of the former the coagulated particles must gather in groups large enough to be seen with the naked eye. Under a microscope the precipitation would undoubtedly be more sensitive. The experiments were carried out in groups of three test tubes. The colloidal solution of copper ferrocyanide was made from very dilute reagents. One gram of potassium ferro-
91
REVERSIBLE PERMEABILITY O F MEMBRANES
cyanide and one gram of copper sulphade, each dissolved in 3000 cc of distilled water, were allowed t o react. A very stable brown colloidal solution was obtained which showed no sign of having precipitated after standing for two weeks. The results are given in Table IV.
TABLE IV Group
Volume
% Methyl alcohol
Time
I
I jcc
0. I O
2
2
I gcc
0.22
2
3 4
I 5cc
0.30
2
I gcc
0.40
2
5
1gcc 15cc I gcc
0.45
2
0.50
2 2
6
7 8 9
I ;cc
0.55 0.60
IjCC
0.65
2
IO
I gcc
0.70
2
IT
IjCC I 5cc
I3 I4 I5 16
I 5cc
0.75 0.80 0.8j
2
I2
0.90 0.9.5 I .oo
2
I.0j
2
I.IO
2
17
18 19
I jcc I gcc
15cc I gcc I gcc I gcc
I8 1.28 I .32 1.36 I.
2
2 2
2 2
2
20
IjCC
21
I gcc
22
I gcc
23
I .41
2
24
1gcc I 5cc
.46
2
25
1gcc
I . go
2
26
I5CC
1.55
2
27
I
gcc I gcc
I'
59 1.64 1.68 1.73
2
I.77
2 2
I 5cc
1.82 I .86 I .g1 1.96 1.96
I 5cc
2.44
2
I 5cc
2.91
2
28
29 30 31 32 33 34 35 36 37 38
IjCC
;cc I 5cc I gcc I gcc I jcc I
IjCC
I
2 2 2
2 2 2
2 2 2 2
days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days days
Degrrr of precipitation
-
-
CHARLES GURCIIOT
92
TABLE I V (continued) Group
39 40 41 42 43 44
45 46 47 48
49 50
51 52
53 54 55 56 57
58 59 60
Vclume
Methyl alcohol
ISCC
3.40
I 5cc
3.85
5cc 15cc I jcc I jcc I 5cc
4.30 4.75
I
IjCC
5cc I 5cc I
I5CC
15cc I 5cc I 5cc I 5cc 51cc I jcc 15cc I5CC
5cc 15cc 15cc I
5.20
5.65 6.10
6.50 7 .oo 7.40
7.80 8.25 10.00
15.00 20.00
25.00 30.00
35.00 40.00
45.00
48.00 50.00
Time
days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 days 2 dyas 2 days 2 days 2 days 2 days 2 days 2 days 2 days
Degree of precipitation
2
?
+ ++ +++ ++++
From the three tubes containing forty percent alcohol 1 4 . 5 of~ ~the colorless supernatant liquid were pipetted off and substituted with the same amount of distilled water. The flocculated copper ferrocyanide was peptized immediately and had not visibly precipitated again after standing one week. This is clearly a case of reversibility without the encumbrance of a membrane. Here it may be argued that reversibility resulted from the increase in surface tension when water was substituted for aqueous alcohol. To say this is to argue post hoc ergo propter hoc. All we know from observation is that reversal was preceded by a surface tension increase. This is very different from saying that reversal was caused by the increase of surface tension. It was pointed out above that the coagulation of copper ferrocyanide sol with about 40% alcohol is not a sensitive test. Yet a method was available whereby the sol could be made just as sensitive, if not more so, as its corresponding membrane to the action of dilute alcohol. But to make the phenomenon more general two other negative sols were used instead, namely sulphur and arsenious sulphide. Both sols were prepared by a method of condensation the detaiIs of which can be found in almost any book of laboratory methods in colloid chemistry. The experimental procedure was to add just enough of some flocculating agent not to precipitate the sol and then to determine the quantity of alcohol-
REVERSIBLE PERMEABILITY O F MEMBRANES
93
ethyl alcohol was used-necessary to flocculate it. The experiments were carried out in test tubes disposed in series of four with several tubes kept as controls, Tables TI-VI.
TABLE V Arsenious Sulphide flocculated with M/8 Sodium Chloride Volume of sol 5cc. Time 24 hours. M/8 XsC1 I cc I . 5cc 2
.occ
2.
;cc
3 . occ 3.5cc
Reaction
None None Marked turbidity Marked turbidity Marked turbidity Granular precipitate
Three tubes to which 1.5cc M/8 NaCl had been added were treated with The fourth was kept as control. After 0.03~ ethyl ~ alcohol (about 0.5%). 2 4 hours the three tubes treated were turbid. The fourth remained clear.
TABLE VI Sulphur flocculated with N/4oo Calcium Chloride Volume of sol gcc. Time 24 hours. N/4oo CaCl? 0 . ICC
0.3cc 0.
gcc
0.7cc 0.9cc
Reaction
None None None Slight turbidity Marked turbidity
Three tubes to which o.;cc N/400 calcium chloride had been added were ~ ~ alcohol. The fourth tube was kept as control. After treated with 0 . 0 2 ethyl standing for 24 hours the three tubes were turbid while the fourth remained clear. The results of these two series of experiments show that under the proper conditions sols can be precipitated by very dilute alcohols. Arsenious sulphide required about 0.5% and sulphur about 0.4% ethyl alcohol. Some qualitative experiments were performed with glucose, sugar and %% glycerin. A dilute sulphur sol was flocculated after 24 hours by glucose and sugar syrups and also by the glycerin solution. Coagulation with Acetic Acid In order to confirm Walden’s results with organic acids experiments were made also with copper ferrocyanide membranes and solutions of acetic acid. Sugar and acetic acid were placed inside the tubes and a solution of blue litmus was placed outside. The litmus turned red after two hours and the outside solution was tested for sugar. The results appear in Table VII.
CHARLES GURCHOT
94
yo Acetic Acid
TABLE VI1 Fehling Precipitate
None Faint trace I .o Trace I .j Moderate The critical concentration seems to be around 0.5% acid. Here again an attempt was made to effect a reversal by treating the membrane with water. N o reversal was obtained. Then, since sodium hydroxide should peptize a negative sol, it was tried as a reversing agent. But here the membranes reversed entirely too much. A fifth-normal solution of NaOH entirely peptized the copper ferrocyanide membranes in six hours. The same result was accomplished when a K/5o NaOH solution was used. Then the membranes were exposed to a N / j KaOH solution for twenty minutes. They turned purplish brown. They were then washed and put aside. Two hours later the gelatine support was detached from the glass and the membranes floated free. Some were then exposed to a N/ j o o NaOH for thirty-six hours. They showed no visible alteration but they remained permcable to sugar. In other words no recovery from coagulation took place. 0.2
0 5
Further Attempts at Reversal Several membranes which had been coagulated previously with methyl alcohol were treated for twenty-four hours with a saturated solution of sodium chloride. The solution was placed on both sides of the membranes so as to preclude dilution by osmosis if pure yTater was put on one side. Some of the membranes showed partial recovery. In other words the Fehling precipitate was much less abundant than before the salt treatment. But on standing for twelve hours they were again tested for sugar permeability. This time the cuprous oxide precipitate was as abundant as it had been before the salt treatment. If there was no large experimental error, the observation is an interesting one perhaps; but as far as indicating tha t reversibility took place it probably means nothing at all. A t this stage it seemed almost a hopeless venture to attempt to reverse coagulated membranes although the thing must be, without a doubt, theoretically possible, On the other hand, looking at it from a different point of view, reversibility might not be so essential. In plant and animal cells the membrane-forming materials might be supplied continually. Recent work has thrown much doubt on this idea. Still if living membranes could reverse their increased permeability and if they availed themselves of the membrane-forming materials only in case of emergency if, at all, the procedure would be far more desirable in the economy of life. I t seemed rather a pity that the latter situation should not prevail. I t probably does in fact, as we shall see later. Effect of the Presence of Membrane-forming Reagents Accordingly, some experiments were tried with copper sulphate on one side and potassium ferrocyanide on the other side of the membrane and of the
95
REVERSIBLE PERMEABILITY O F MEMBRANES
same concentration as the reagents used in making the membranes. Controls, without any of the reagents on either side, were used in every case. Although the contents on the inside of the tubes varied, the outside solution did not vary from one experiment to another. I n every case potassium ferrocyanide and distilled water only were present outside the membranes. The presence of potassium ferrocyanide interfered, of course, with the detection of sugar. The hydrochloric acid which was added to hydrolyze the sucrose reacted with potassium ferrocyanide t o form ferrocyanic acid. This came down as a white precipitate and in addition to using up the HC1 obscured any other precipitate which might form, As a result the procedure summarized below was adopted. It was found to work perfectly well on known samples: I. Precipitated K4Fe(CK)6with slightly more than half its volume 0.04 molar copper sulphate. 2. Disregarded brown precipitate, added 2 drops conc. HC1 and boiled. 3. Added 2cc of a saturated solution of XaHC03, boiled off COZ excess. 4. Allowed copper ferrocyanide precipitate to settle. 5. Pipetted clear supernatant liquid. 6. Boiled with Fehling’s solution in the usual way. The results of the experiments are given in Table VIII.
TABLE VI11 Inside CH,OH
KaC1
CaCI2
Sugar
0 . o2M
0.02M
3%
1%
5%
2%
o2M
0 . o2M
0 . o2M
0 . o2M
0.02M
0 . o2M
0 . o2M
o2M
0.
0.
2%
10%
5%
1% 2%
2.5%
0 . o2M
0 . o2M
5%
.25%
1%
25%
1.25%
2%
5%
1%
o.ozM
0 . o2M
0 . o2M
0.02M
0 . o2M
0.02M
0 . o2M
0 . o2M
2%
1%
1%
0.02M
0 . o2M
5%
5%
2%
12.5%
5%
2%
2%
Time Reaction
48 hrs
hrs hrs 48 hrs 24 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs 48 hrs I O hrs 72
24
-
-
-
-
t-
Only the last experiment, where no membrane-forming reagents were used, showed the membrane to have coagulated, A11 other tests showed negative results, even though in the case of sodium chloride the salt was found outside the membrane. Not so with calcium chloride, Cupric chloride was used instead of copper sulphate to prevent the formation of calcium sulphate.
96
CHARLES GURCHOT
Critical Concentrations of Alcohols and Reversal of Coagulation The next part of this investigation was carried out during cold weather when the temperature of the heated laboratory rose to 26'C. Indeed it reached 27OC. more than once. This circumstance was very unfortunate in one way, but it proved very fortunate in another way. First of all, the copper ferrocyanide membranes did not work properly. The yield was very small. Sometimes every new membrane was permeable to sugar before having been used at all. It was thought that the gelatine softened and impaired the membrane support. Various schemes were tried to find some twenty-seven degree-proof support for the membrane but none was found satisfactory. Among other things agar-agar was tried. Owing to the fact that agar is soluble in water only a t about 85OC. it needs no tanning treatment and would make an ideal support if it were not that it shrinks on cooling and loosens itself from the glass tubes. This loosening did not take place however until the copper ferrocyanide membrane was deposited. This loosened membrane was, of course, useless for diffusion experiments, but its structure could be examined in detail. It was found that the membrane had formed about I.5mm below the inside surface, and that the agar above the membrane was colored brown for a distance of about Imm. This was clearly a case of peptization of copper ferrocyanide in agar and as far as the membrane itself was concerned it appeared intact. The peptizing agent could be only copper sulphate. On the potassium ferrocyanide side the membrane was not peptized visibly. There is nothing strange in this for the silver halides are peptized by silver nitrate and the corresponding potassium halide. Here was a suggestion for one more attempt at reversibility. But in the mean time stable membranes had to be made. This was finally done by using a 15% gelatine solution to begin with, forming the membranes, and keeping them at all times, in low temperature thermostats. Several were made, tap water being used as the cooling medium. All winter the temperature in the thermostats varied between 6 and 8 degrees centigrade. Once or twice it rose to tendegrees but never higher. Another possibility not to be overlooked was that copper ferrocyanide membranes might be coagulated by pure water at comparatively low temperatures. But before heat coagulation was investigated in addition to a possible reversal with copper sulphate it was necessary to determine the critical concentration for several of the alcohols used by Czapek and see what relation they bore to the critical concentrations which he obtained as a result of his experiments on exosmosis. The four alcohols chosen were methyl, ethyl, propyl and amyl. I n addition to this, since it was found that sodium chloride coagulated copper ferrocyanide its critical concentration should also be determined and compared with that of potassium chloride and calcium chloride; three salts which play a very important part in the metabolism of living cells,Table IX.
97
REVERSIBLE PERMEABILITY OF MEMBRANES
TABLE IT; Concentration
Sugar test
Estimate of critical conrentrntion from appearances of ppts.
1.5%
Methyl Alcohol negative positive
Below
0.4% 0.6%
Ethyl Alcohol negative positive
Between .5-.
Propyl Alcohol negative positive
About
.2
About
2
0.1%
I.
5yo
6%
%
Amyl Alcohol negative negative negative positive Sodium Chloride negative negative positive
%3
Potassium Chloride negative negative positive
About, 2y0
Calcium Chloride negative positive positive
Below rci;i
It is interesting to note that no calcium was found in the outside water for the 0 . 5 % tube. A trace of calcium chloride was found for the 1% tube; and the 1.5% tube gave a very decided test for both calcium and chloride in the outside water. The reversibility experiments with copper sulphate were much more successful than the previous attempts. The results were decisive except perhaps in the case of ethyl alcohol where a more concentrated copper sulphate solution had to be used in order to effect a reversal. Three tubes were used in every case for each alcohol. The sugar tests were negative, Table X. There are, of course, many experiments which might have been tried on the basis of the results given above. Time did not permit it, however. The peptizing action of copper sulphate on precipitated copper ferrocyanide sols
98
CHARLES GURCHOT
TABLEX Colzgulnting agent
Concentration of copper sulphate
Methyl alcohol Ethyl alcohol Ethyl alcohol Propyl alcohol Amyl alcohol
Number of tubes which recovered
0.02M
3
o2M
0
o.20M 0 . o2M
3
0 . o2M
3
0.
2
might have been tried as an analogy to the peptization of silver halide precipitates. Also, the addition of copper sulphate to sodium chloride solution should raise the latter's coagulating concentration. The same should be true of copper sulphate and alcohols. Another interesting suggestion was furnished from work done with colloidal solutions of ferric oxide (Briggs and Vannoy 192 5). It was found that concentrated solutions of calcium chloride (above 30%) peptized ferric oxide formed from ferric chloride and ammonium hydroxide, producing a negative sol. But when coagulated copper ferrocyanide membranes were treated wjth syrupy calcium chloride solution (707~) not only did they not recover, but after the treatment they allowed far more sugar to go through than before.
Coagulation by Heat For these experiments a small oven was used jacketed with running water and heated with a bunsen flame. The temperature varied within one degree. The net result obtained was that copper ferrocyanide membranes were coprobably 24OC. The figulated by pure water between 23 and 24'C.-more futility of working with such membranes at room temperature of 2 6 O C . becomes apparent at once. There is no doubt, of course, that when the temperature of the water is raiPed its surface tension is lowered; and coagulation of copper ferrocyanide a t 23 or 24 degrees may have been brought about by the surface tension lowering. But it is also true that adsorption decreases with rise of temperature, that consequently the film of adsorbed water around the membrane particles is not as thick as it was before which is the same as saying that effectual pores may be formed allowing such things as sugar to go through which would otherwise be retained by virtue of the negative adsorption exhibited by copper ferrocyanide towards sugar. This is all the more convincing when we consider the lack of agreement between the surface tensions of several alcohols and water a t 23.5'C, Table XI.
TABLE XI Reagrnt
Methyl alc. Ethyl alc. Amyl alc. Water 2 3 . 5 ' Water 8'
Concentration
1.5% 54% .2%
Surface tension
6 6 . 5 dynes 67 dynes 6 8 . 5 dynes 7 I . 8 dynes 7 4 . 5 dynes
% Lowering of surfacp tension against HOH at 8°C: 11% 10%
8% 4% 0%
REVERSIBLE PERMEABILITY O F MEMBRANES
99
Conclusions Before discussing the relation between the experimental results and the behavior of cell membranes we must take up the question of coagulation from the point of view of semi-permeable membranes in general of the type represented by copper ferrocyanide. There is no question about the external appearance of copper ferrocyanide membranes deposited in gelatin. Microscopic examination undertaken by means of objectives of high resolving power (N. A. 1.4, Zeiss Apochromat) showed beyond a doubt that the membrane is granular in character. Separate grains or groups of them are present and make up the entire area. Their size could not be measured, of course, owing to the presence of overlapping diffraction halos; and it is difficult to see how Tinker1 in 1916 calculated the size of copper ferrocyanide particles by means of the wretched photographs which he published. Since copper ferrocyanide forms a hydrosol each particle or group must possess a layer of adsorbed water. When alcohol is added there are three possibilities: First, coagulation is brought about by a decrease in surface tension. This explanation is tenable only if we consider the copper ferrocyanide film to be a continuous membrane like rubber. We know that this is not the case. But if we consider the membrane to be granular it is not clear how surface tension decrease can be a factor in coagulation. The particles should tend to break up into still smaller ones and in the end a finer grained membrane would result which if anything should be less permeable than before. Second, if coagulation is due to selective adsorption it may involve the entire alcohol molecule or the positive radical. But when a sol coagulates it does so because the charge on each particle which keeps all particles apart is reduced or neutralized and the part,iclesagglomerate. In other words the adsorption of alcohol by a negatively charged sol like copper ferrocyanide must entail a reduction or neutralization of its negative charge. The electrically neutral aggregates can unite to form larger units and finally one large coagulum will result. The difference between such a final aggregation and the original one is that its area is smaller, it is more compact and water, both adsorbed and not, will be present around it. At no time then is coagulation reversed as in the case of decreased surface tension. The last possibility is the removal of water of hydration from the supposedly coninuous hydrated copper ferrocyanide membrane with consequent shrinkage tnd the formation of cracks which would render the membrane permeable to ,hings to which it is ordinarily impermeable. It was this consideration which irompted Walden and others to retain the membrane-forming reagents on loth sides of the copper ferrocyanide. But if coagulation were indeed a iuestion of the removal of water of hydration-the fact that copper ferroayanide is granular alone throws a damper over the hydration theory-the ‘racks formed in its substance could not be obliterated by peptizing with cop)er sulphate. It may be argued that copper sulphate did not peptize the memwane but that instead it reacted with adsorbed potassium ferrocyanide which ould not be washed out and the cracks were healed by the reprecipitation of Proc. Roy. Soc., 92A, 357 (1916).
IO0
CHARLES GURCHOT
copper ferrocyanide. The microscopic examination of the membrane in agar which showed the presence of peptized membrane material militates against the strength of this argument in addition to the fact that we know such things as silver halides to be peptized by their own reagents. Unless something has been left out of consideration we can be fairly certain so far that coagulation is a question of selective adsorption, washing out the alcohol with water will not necessarily peptize the membrane and thereby make it impermeable to sugar once more. The coagulated particles merely take collectively their film of adsorbed water and the spaces remain through which sugar can pass. The rigidity of the gelatine support is alone responsible for this because we have seen that in test tubes a coagulated copper ferrocyanide sol could be repeptized by the addition of water. In the latter case the addition of water was tantamount to a mechanical disintegration for the flocculated copper ferrocyanide was still in a very fine state of subdivision. The amount of alcohol remaining after peptization of the sol was 1.3% which is the amount just necessary to make a membrane permeable to sugar. But, as it was suggested before, the degree of coagulation could not be appreciated possibly since 40% alcohol was necessary to make the phenomenon visible in a test tube to the naked eye. Then there is the question of coagulation by heat. If the membrane were a continuous one it is difficult to see how a decrease in the surface tension of about one dyne produced by an increase of a few degrees-it must be remembered that at 2oOC. successful experiments were performed-should make it permeable to sugar, although we must admit that the thing would not be impossible. But if the phenomenon is connected with surface tension then there must be a critical tension when a continuout membrane becomes discontinuous-forms drops in other words. The evi. dence given above shows that there is not. Alcohols acted a t about 90% the original tension, water a t 96%. If we must postulate a toxic action certainlj we cannot blame it on the water. The alcohols cannot be blamed either be cause the discrepancy between methyl, ethyl, normal propyl, iso-propyl, butyl iso-butyl, amyl and allyl and tertiary butyl is attributed to a toxic action 01 the part of the latter two. But, after all, the microscopic evidence is unequi vocal enough. We cannot say that the copper ferrocyanide membrane operates like : sieve because such a statement is only a small part of the story. It may ac as a sieve for sodium and potassium chloride only under certain condition because if the salts are present in dilute enough solution they will go througl the membrane without coagulating it. This can be easily explained. If t h adsorption isotherm for chloride ion in the presence of sodium ion, for exampk reaches a constant value much before the isotherm for sodium ion in th presence of chloride, there must be a concentration for which both ions will b adsorbed equally. Since sodium chloride is soluble in water it will dissolve i the adsorbed water layer, under such conditions, and pass through the men brane without coagulating it. At higher concentrations the sodium ion will b adsorbed more and more whereas the chloride ion will not. As a result ther
REVERSIBLE PERMEABILITY O F I\IEMBRANES
IO1
must come a point when the membrane will no longer be negative and coagulation will ensue. From this point on sugar will pass through the membrane, provided the membrane-forming reagents are absent. Sugar molecules which are adsorbed negatively will not go through therefore, until effective pores have been formed, This will not happen as long as both the membrane-forming reagents are present. The case of calcium chloride is interesting. TTaldenl found that sometimes it did go through and a t other times it did not. This happened when he used copper acetate one on side and potassium ferrocyanide on the other. Here Walden must have been using calcium chloride solutions of somewhere near the critical concentration below which it would not go through copper ferrocyanide when the membrane-forming reagents were present. I n fact earlier in his paper Walden says that his diffusing solutions were about normal. But finally he succeeded in preventing his calcium chloride solutions from going through when, instead of copper acetate, he used copper nitrate on one side of the membrane, When this is correlated with the results obtained from experiments on reversibility with copper sulphate, described earlier in the present paper the explanation is easy, Copper ferrocyanide probably adsorbs the nitrate ion more readily than it does the acetate ion. Consequently the membrane is kept negative, and for the same concentration of calcium chloride in both cases the latter will not go through in the presence of copper nitrate. With calcjum chloride the adsorption isotherm reaches its constant value probably at such a low concentration that calcium chloride will pass through the membrane in negligible amounts only without coagulating it. In other words calcium chloride will not pass through the membrane until it coagulates it. And so we find that as soon as we can obtain a test for calcium chloride in the outside water sugar is present there also, Finally we can say that a substance in solution will go through a membrane by osmosis when like sodium chloride it will go through without producing coagulation. When a membrane allows any true solution to go through including those solutions whose solutes are adsorbed negatively, then it is acting as a dialyzing membrane. It is understood, of course, that a solution which passes through by osmosis may under proper conditions-namely, after the membrane has coagulated-go through by diffusion or capillarity as through a dialyzing membrane. Calcium chloride does not necessarily fall out of the first class because even though both its ions are adsorbed equally, only a t extremely low concentrations very dilute solutions will go through appreciably after a long enough time has elapsedwithout the membrane having been coagulated. And so sugar will go through copper ferrocyanide not because the latter is an ultrafilter, but because it has been converted into one through selective adsorption of cations and consequent coagulation. When a substance is brought in contact with a membrane in this condition and for which it possesses greater adsorption for the anion than for the cation the situation is reversed, the pores are closed because the copper ferrocyanide is Z. physik. Chem. 10, 699 (1892).
I02
CHARLES GUR CHOT
peptized and it is no longer permeable to sugar. Such a membrane represents therefore a dynamic system. Its structure and its behavior must depend on the character of the substance which is trying to pass through. All this is very suggestive when we consider what takes place in the case of living cells. When Collanderl contends that protoplasm probably acts as an ultra-filter toward substances which are not soluble in lipoids he does not apparently take into consideration the fact that cells are both permeable and impermeable ’ for the same substance. I n addition to this he assumes a sort of continuous lipoid film with holes in it. This is going beyond the facts known a t present. Collander also takes it for granted that cells have present at all times on either side the membrane-forming reagents. KOsuch simple assumption is adequate. It is not known how the membrane is formed. It may be kept intact by the constant accumulation on one side and dissipation on the other of substances which lower the surface tension of a liquid as they concentrate at the interface between the cell and the external medium; or by secretion or by a slow process of precipitation2. “A feature of the protoplasm of many cells is the existence of a more or less differentiated cortex. When the cortex is well differentiated, it is an appreciable zone of protoplasm which is more solid than the interior. This is specially well developed in many Protozoa. Paramoecium, for example, possesses a firm cortex, the ectoplasm, of a uniform thickness over the body of theorganism. It is a structural modification which cannot be easily, if at all, repaired by the more fluid endoplasm. In Paramoecium it is an elastic jellylike wall which is under a certain tension due to internal turgor. This can be shown by tearing the ectoplasm with a micro needle. The fluid interior then pours out, while the torn edges of the ectoplasm curl in, and the entire animal shrinks in size. If the edges of the tear meet they unite and further outflow of the endoplasm ceases, otherwise all the endoplasm flows out and the ectoplasm soon disintegrates. The endoplasm as it pours out into the surrounding water occasionally forms a delicate surface film which maintains the integrity of the extruded mass. Usually, however, the endoplasm becomes entirely dissipated and disintegrates. I n some species, e.g. Paramoecium bursaria, the torn surface very readily forms a surface film which frequently persists. The integrity of the cell is thereby maintained. In the few experiments that have been made on this form, the endoplasmic surface film is apparently unable to regenerate a differentiated ectoplasmic layer. Complete recovery occurs by a gradual contraction of the animal until the ectoplasmic edges bordering the ectoplasmic surface film, meet and unite”. Again “The surface film of blood cells and of tissue cells isolated in blood plasma or serum is extremely susceptible to mechanical injury unless special precautions are taken to maintain conditions as normal as possible. The human red blood corpuscle furnishes an interesting example of a rapid breakdown of the surface film. When the surface film is punctured with a very fine glass micro needle the 1 2
Kolloidchem. Beihefte, 20, 273 (1925). Chambers: “Genera1 Cytology”, 254, 255, 257 (1924).
REVERSIBLE PERMEABILITY O F MEMBRANES
103
hemoglobin immediately diffuses out all over the cell and leaves behind a transparent glutinous mass which can be torn into strands”. From what has just been said it is evident that living membranes are left pretty much to their own resources, as it were, to manifest their phenomena of semi-permeability. And, therefore, if the copper ferrocyanide membrane is to exhibit analogous properties it must be used so as to imitate most nearly the conditions imposed upon living membranes. The first of these conditions requires the absence of the membrane-forming reagents. This condition Collander did not fulfill in his experiments. I n addition to this the statement that the living membrane is probably an ultra-filter needs further correction. Collander has not taken the trouble, apparently, to differentiate between a semi-permeable membrane and an ultra-filter. To be sure he is not the only one. An ultra-filter is something which has been taken more or less for granted, and as a result no really definite idea has been in vogue. Accordingly Bancroftl has formulated the definition that “an ultra-filter js essentially a porous membrane which will, by hypothesis, never become a semi-permeable membrane unless there is a strong negative adsorption-preferential adsorption of the solvent.” “The conclusion that a substance which can be filtered out through an ultra-filter is in colloidal solution and not in true solution rests on the explicit assumption that any gas or vapor will go through any pore through which any other gas or vapor will go”. According to this idea-with which, after all, Collander cannot be confronted-everything in the cell which is in solution will ooze out and everything which is in solution will go in. The composition of the inside of the cell, as far as solutes go, will be identical with that outside. This, of course, is impossible. The simple ultra-filter idea must be revised. On the other hand coagulation of the membrane and a reversal of the same explain a great many things, even though the structural character of the membrane is still a puzzle. The objection to’ Overton’s lipoid theory2 was that water-soluble substances like sugar could not penetrate a lipoid membrane. If however we assume the presence of a granular lipoid membrane analogous to copper ferrocyanide, in other words with each solid fat particle, or group of particles, surrounded with an adsorbed film of water supported in a separate pellicle, which is known to be present, we have essentially the same sort of thing as the copper ferrocyanide membrane and there seems to be no reason why, should such a membrane exist actually, it should not exhibit coagulation changes when necessary and allow e.g. sugar to enter the cell a t one time and not a t another. We have still to account however for the entrance of lipoid solvents through such a membrane. This is not at all a hopeless endeavor. Mention was made above of a film of adsorbed water surrounding the lipoid particles. It need not be that necessarily. The film could be a solution possessing properties analogous to a pyridine solution. I n other words it might dissolve substances other than and in addition to lipoid solvents. In this way 2
J. Phys. Chem. 29, 966 (1925). Jahrbuch wiss. Botanik, 34, 669 (1899).
104
CHARLES GURCIIO'L'
the lipoid particles would be available as carriers for substances like ether and chloroform. Such a membrane would have the advantage of manifesting osmotic pressure, unlike an emulsion, and also to exhibit permeability changes in localized areas of the cell surface. It must be understood that only a very generalized picture of cell membrane is attempted. Undoubtedly membranes must differ with the functions of the particular organs of which they form a part. It may be objected that a substance like pyridine which may dissolve lipoid solvents may also dissolve the lipoid portion of the membrane and so destroy it. This need not follow a t all. Pyridine itself is soluble in rubber, but it has been used with rubber membranes successfully by Kzhlenberg without the rubber being dama,ged even in boiling pyridine. On the other hand if the idea of a discontinuous membrane-that is a fine grained onewith adsorbed water around the particles is not an attractive one we can postulate the existence of a continuous lipoid film of some sort which acts ordinarily as a semi-permeable membrane allowing only such things to go through which dissolve in it. A change in the surface tension of the surrounding medium might coagulate such a membrane into solid or semi-solid drops and allow water-soluble substances to pass through. Such a membrane could be reversed by changing the surface tension of the surrounding medium perhaps in addition to the extraction of the coagulating agent from between the particles by the presence of a suitable solvent or reagent. The coagulating agent might be a water soluble soap resulting from the reaction between sodium chloride and some fatty compound. This soap would reduce the surface tension of the solid film and pores would be formed between the solid drops in the membrane. The presence of calcium chloride would form a water-insoluble inert calcium soap which would dissolve in the membrane and subsequently would be decomposed by the cell after an increase in the surface tension of the external medium reversed the membrane and made it once more continuous. Clowes was not far away from such an explanation but he insisted that the cell must be an emulsion.
For the membrane suggested at first, antagonism can exist between the substances which will tend to coagulate the membrane and those which will peptize it. In the case of copper ferrocyanide the antagonism established was between alcohol or an inorganic salt on the one hand and copper sulphate on the other. There are undoubtedly other substances besides copper sulphate which will accomplish the same thing. The object of the investigation was not so much to find what would peptize the coagulated membrane as to make sure that the re-peptization could be accomplished at all. The exact character of such antagonism as sodium-calcium must be left for future investigation when a membrane more nearly akin to a living one will be made. We know that after death the cell membrane behaves as ordinary dialyzer. The phenomenon is probably a question of permanent and irreversible coagulation. Experiments with acetic acid suggest the cause to be increased acidity of living cells after death owing to the accumulation of lactic acid.
REVERSIBLE PERMEABILITY O F MEMBRANES
10.5
There is no doubt that coagulation and reversibility of cell membranes suggest an interesting line of attack for many a problem in physiology and pathology. Whether the analogy, which has been drawn, in this series of investigations, between an artificial membrane and ectoplasm is warranted time alone will tell.
Summary I. Copper ferrocyanide membranes are coagulated by low concentrations of methyl alcohol, acetic acid, sodium chloride, potassium chloride and calcium chloride, thereby becoming permeable to sugar. A copper ferrocyanide sol is flocculated by aqueous alcohol, whereas 2. alkalies and water peptize a copper ferrocyanide gel. 3 . The coagulation of copper ferrocyanide is caused by selective adsorption rather than by decrease in surface tension. 4. Copper ferrocyanide forms a granular membrane between those particles adsorbed water is present which fills the spaces more or less completely. 5 . Copper ferrocyanide membranes are coagulated by water at a temperature of about 2 4 O C . 6 . The coagulation of copper ferrocyanide is reversible. 7 . Bartlow’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. 8. Intermittent permeability in living membranes is probably also a question of reversible coagulation, This problem was suggested by Professor W. D. Bancroft to whom the writer is greatly indebted for invaluable criticism and suggestions.
Cornell Unwersziy.
