INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON SOME PHYSICAL PROPERTIES OF COLLOIDS AND OF CRYSTALLOIDS

1. When a 1 per cent solution of a metal gelatinate, e.g. Na gelatinate, of pH = 8.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate which can be measured by the rise of the level of the liquid in a manometer. When to such a solution alkali or neutral salt is added the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. This depressing effect of the addition of alkali and neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. 2. When a neutral M/256 solution of a salt with monovalent cation (e.g. Na₂SO₄ or K₄Fe(CN)₆, etc.) is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain initial rate. When to such a solution alkali or neutral salt is added, the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. The depressing effect of the addition of alkali or neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. The membranes used in these experiments were not treated with gelatin. 3. It can be shown that water diffuses through the collodion membrane in the form of positively charged particles under the conditions mentioned in (1) and (2). In the case of diffusion of water into a neutral solution of a salt with monovalent or bivalent cation the effect of the addition of electrolyte on the rate of diffusion can be explained on the basis of the influence of the ions on the electrification and the rate of diffusion of electrified particles of water. Since the influence of the addition of electrolyte seems to be the same in the case of solutions of metal gelatinate, the question arises whether this influence of the addition of electrolyte cannot also be explained in the same way, and, if this be true, the further question can be raised whether this depressing effect necessarily depends upon the colloidal character of the gelatin solution, or whether we are not dealing in both cases with the same property of matter; namely, the influence of ions on the electrification and rate of diffusion of water through a membrane. 4. It can be shown that the curve representing the influence of the concentration of electrolyte on the initial rate of diffusion of water from solvent into the solution through the membrane is similar to the curve representing the permanent osmotic pressure of the gelatin solution. The question which has been raised in (3) should then apply also to the influence of the concentration of ions upon the osmotic pressure and perhaps other physical properties of gelatin which depend in a similar way upon the concentration of electrolyte added; e.g., swelling. 5. When a 1 per cent solution of a gelatin-acid salt, e.g. gelatin chloride, of pH 3.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate. When to such a solution acid or neutral salt is added—taking care in the latter case that the pH is not altered—the initial rate with which water will diffuse into the solution is diminished and the more so the more acid or salt is added. Water diffuses into a gelatin chloride solution through a collodion membrane in the form of negatively charged particles. 6. When we replace the gelatin-acid salt by a crystalloidal salt, which causes the water to diffuse through the collodion membrane in the form of negatively charged particles, e.g. M/512 Al₂Cl₆, we find that the addition of acid or of neutral salt will diminish the initial rate with which water diffuses into the M/512 solution of Al₂Cl₆, in a similar way as it does in the case of a solution of a gelatin-acid salt.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

THE ORIGIN OF THE POTENTIAL DIFFERENCES RESPONSIBLE FOR ANOMALOUS OSMOSIS

1. Collodion bags coated with gelatin on the inside were filled with a M/256 solution of neutral salt (e.g., NaCl, CaCl₂, CeCl₃, or Na₂SO₄) made up in various concentrations of HNO₃ (varying from N/50,000 to N/100). Each collodion bag was put into an HNO₃ solution of the same concentration as that inside the bag but containing no salt. In this case water diffuses from the outside solution (containing no salt) into the inside solution (containing the salt) with a relative initial velocity which can be expressed by the following rules: (a) Water diffuses into the salt solution as if the particles of water were negatively charged and as if they were attracted by the cation and repelled by the anion of the salt with a force increasing with the valency of the ion. (b) The initial rate of the diffusion of water is a minimum at the hydrogen ion concentration of about N/50,000 HCl (pH 4.7, which is the point at which gelatin is not ionized), rises with increasing hydrogen ion concentration until it reaches a maximum and then diminishes again with a further rise in the initial hydrogen ion concentration. 2. The potential differences between the salt solution and the outside solution (originally free from salt) were measured after the diffusion had been going on for 1 hour; and when these values were plotted as ordinates over the original pH as abscissae, the curves obtained were found to be similar to the osmotic rate curves. This confirms the view expressed by Girard) Bernstein, Bartell, and Freundlich that these cases of anomalous osmosis are in reality cases of electrical endosmose where the driving force is a P.D. between the opposite sides of the membrane. 3. The question arose as to the origin of these P. D. and it was found that the P.D. has apparently a double origin. Certain features of the P.D. curve, such as the rise and fall with varying pH, seem to be the consequence of a Donnan equilibrium which leads to some of the free HNO₃ being forced from the solution containing salt into the outside solution containing no (or less) salt. This difference of the concentration of HNO₃, on the opposite sides of the membrane leads to a P.D. which in conformity with Nernst''s theory of concentration cells should be equal to 58 x (pH inside minus pH outside) millivolts at 18°C. The curves of the values of (pH inside minus pH outside) when plotted as ordinates over the original pH as abscissae lead to curves resembling those for the P. D. in regard to location of minimum and maximum. 4. A second source of the P.D. seems to be diffusion potentials, which exist even if no membranes are present and which seem to be responsible for the fact that the rate of diffusion of negatively charged water into the salt solution increases with the valency of the cation and diminishes with the valency of the anion of the salt. 5. The experiments suggest the possibility that the establishment of a Donnan equilibrium between membrane and solution is one of the factors determining the Helmholtzian electrical double layer, at least in the conditions of our experiments.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS

1. It has been shown in previous publications that when solutions of different concentrations of salts are separated by collodion-gelatin membranes from water, electrical forces participate in addition to osmotic forces in the transport of water from the side of the water to that of the solution. When the hydrogen ion concentration of the salt solution and of the water on the other side of the membrane is the same and if both are on the acid side of the isoelectric point of gelatin (e.g. pH 3.0), the electrical transport of water increases with the valency of the cation and inversely with the valency of the anion of the salt in solution. Moreover, the electrical transport of water increases at first with increasing concentration of the solution until a maximum is reached at a concentration of about M/32, when upon further increase of the concentration of the salt solution the transport diminishes until a concentration of about M/4 is reached, when a second rise begins, which is exclusively or preeminently the expression of osmotic forces and therefore needs no further discussion. 2. It is shown that the increase in the height of the transport curves with increase in the valency of the cation and inversely with the increase in the valency of the anion is due to the influence of the salt on the P.D. (E) across the membrane, the positive charge of the solution increasing in the same way with the valency of the ions mentioned. This effect on the P.D. increases with increasing concentration of the solution and is partly, if not essentially, the result of diffusion potentials. 3. The drop in the transport curves is, however, due to the influence of the salts on the P.D. (ε) between the liquid inside the pores of the gelatin membrane and the gelatin walls of the pores. According to the Donnan equilibrium the liquid inside the pores must be negatively charged at pH 3.0 and this charge is diminished the higher the concentration of the salt. Since the electrical transport is in proportion to the product of E x ε and since the augmenting action of the salt on E begins at lower concentrations than the depressing action on ε, it follows that the electrical transport of water must at first rise with increasing concentration of the salt and then drop. 4. If the Donnan equilibrium is the sole cause for the P.D. (ε) between solid gelatin and watery solution the transport of water through collodion-gelatin membranes from water to salt solution should be determined purely by osmotic forces when water, gelatin, and salt solution have the hydrogen ion concentration of the isoelectric point of gelatin (pH = 4.7). It is shown that this is practically the case when solutions of LiCl, NaCl, KCl, MgCl₂, CaCl₂, BaCl₂, Na₂SO₄, MgSO₄ are separated by collodion-gelatin membranes from water; that, however, when the salt has a trivalent (or tetravalent?) cation or a tetravalent anion a P.D. between solid isoelectric gelatin and water is produced in which the wall assumes the sign of charge of the polyvalent ion. 5. It is suggested that the salts with trivalent cation, e.g. Ce(NO₃)₃, form loose compounds with isoelectric gelatin which dissociate electrolytically into positively charged complex gelatin-Ce ions and negatively charged NO₃ ions, and that the salts of Na₄Fe(CN)₆ form loose compounds with isoelectric gelatin which dissociate electrolytically into negatively charged complex gelatin-Fe(CN)₆ ions and positively charged Na ions. The Donnan equilibrium resulting from this ionization would in that case be the cause of the charge of the membrane.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : II. OSMOTIC PRESSURE.

1. It had been shown in previous publications that the osmotic pressure of a 1 per cent solution of a protein-acid salt varies in a characteristic way with the hydrogen ion concentration of the solution, the osmotic pressure having a minimum at the isoelectric point, rising steeply with a decrease in pH until a maximum is reached at pH of 3.4 or 3.5 (in the case of gelatin and crystalline egg albumin), this maximum being followed by a steep drop in the osmotic pressure with a further decrease in the pH of the gelatin or albumin solution. In this paper it is shown that (aside from two minor discrepancies) we can calculate this effect of the pH on the osmotic pressure of a protein-acid salt by assuming that the pH effect is due to that unequal distribution of crystalloidal ions (in particular free acid) on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 2. It had been shown in preceding papers that only the valency but not the nature of the ion (aside from its valency) with which a protein is in combination has any effect upon the osmotic pressure of the solution of the protein; and that the osmotic pressure of a gelatin-acid salt with a monovalent anion (e.g. Cl, NO₃, acetate, H₂PO₄, HC₂O₄, etc.) is about twice or perhaps a trifle more than twice as high as the osmotic pressure of gelatin sulfate where the anion is bivalent; assuming that the pH and gelatin concentrations of all the solutions are the same. It is shown in this paper that we can calculate with a fair degree of accuracy this valency effect on the assumption that it is due to the influence of the valency of the anion of a gelatin-acid salt on that relative distribution of the free acid on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 3. The curves of the observed values of the osmotic pressure show two constant minor deviations from the curves of the calculated osmotic pressure. One of these deviations consists in the fact that the values of the ascending branch of the calculated curves are lower than the corresponding values in the curves for the observed osmotic pressure, and the other deviation consists in the fact that the drop in the curves of calculated values occurs at a lower pH than the drop in the curves of the observed values.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS : II. INFLUENCE OF THE RADIUS OF THE ION.

1. When solutions of KCl, NaCl, or LiCl are separated from water without salt by a collodion-gelatin membrane and when the pH of both salt solution and water are on the acid side of the isoelectric point of gelatin, water diffuses from the side of pure water into the salt solution at a rate increasing inversely with the radius of the cations. 2. The adsorption theory would lead us to assume that this influence of the cations is due to an increase of the P.D. between the liquid and the membrane inside the pores of the gelatin film of the membrane, but direct measurements of this P.D. contradict such an assumption, since they show that the influence of the three salts on this P.D. is identical at pH 3.0. 3. It is found, however, that the P.D. across the membrane is affected in a similar way by the three cations as is the transport of water through the membrane. 4. This P.D. across the membrane varies inversely as the relative mobility of the three cations which suggests that the influence of the three cations on the diffusion of liquid through the membrane is partly if not essentially due to a diffusion potential.     

IONIZING INFLUENCE OF SALTS WITH TRIVALENT AND TETRAVALENT IONS ON CRYSTALLINE EGG ALBUMIN AT THE ISOELECTRIC POINT

1. While crystalline egg albumin is highly soluble in water at low temperature at the pH of its isoelectric point, it is coagulated by heating. It has long been known that this coagulation can be prevented by adding either acid or alkali, whereby the protein is ionized. 2. It is shown in this paper that salts with trivalent or tetravalent ions, e.g. LaCl₃ or Na₄Fe(CN)₆, are also able to prevent the heat coagulation of albumin at the isoelectric point (i.e. pH 4.8), while salts with a divalent ion, e.g. CaCl₂, BaCl₄, Na₂SO₄, or salts like NaCl, have no such effect. 3. This is in harmony with the fact shown in a preceding paper that salts with trivalent or tetravalent ions can cause the ionization of proteins at its isoelectric point and thus give rise to a membrane potential between micellæ of isoelectric protein and surrounding aqueous solution, while the above mentioned salts with divalent and monovalent ions have apparently no such effect.     

ON THE CAUSE OF THE INFLUENCE OF IONS ON THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES. I

1. In three previous publications it had been shown that electrolytes influence the rate of diffusion of pure water through a collodion membrane into a solution in three different ways, which can be understood on the assumption of an electrification of the water or the watery phase at the boundary of the membrane; namely, (a) While the watery phase in contact with collodion is generally positively electrified, it happens that, when the membrane has received a treatment with a protein, the presence of hydrogen ions and of simple cations with a valency of three or above (beyond a certain concentration) causes the watery phase of the double layer at the boundary of membrane and solution to be negatively charged. (b) When pure water is separated from a solution by a collodion membrane, the initial rate of diffusion of water into a solution is accelerated by the ion with the opposite sign of charge and retarded by the ion with the same sign of charge as that of the water, both effects increasing with the valency of the ion and a second constitutional quantity of the ion which is still to be defined. (c) The relative influence of the oppositely charged ions, mentioned in (b), is not the same for all concentrations of electrolytes. For lower concentrations the influence of that ion usually prevails which has the opposite sign of charge from that of the watery phase of the double layer; while in higher concentrations the influence of that ion begins to prevail which has the same sign of charge as that of the watery phase of the double layer. For a number of solutions the turning point lies at a molecular concentration of about M/256 or M/512. In concentrations of M/8 or above the influence of the electrical charges of ions mentioned in (b) or (c) seems to become less noticeable or to disappear entirely. 2. It is shown in this paper that in electrical endosmose through a collodion membrane the influence of electrolytes on the rate of transport of liquids is the same as in free osmosis. Since the influence of electrolytes on the rate of transport in electrical endosmose must be ascribed to their influence on the quantity of electrical charge on the unit area of the membrane, we must conclude that the same explanation holds for the influence of electrolytes on the rate of transport of water into a solution through a collodion membrane in the case of free osmosis. 3. We may, therefore, conclude, that when pure water is separated from a solution of an electrolyte by a collodion membrane, the rate of diffusion of water into the solution by free osmosis is accelerated by the ion with the opposite sign of charge as that of the watery phase of the double layer, because this ion increases the quantity of charge on the unit area on the solution side of the membrane; and that the rate of diffusion of water is retarded by the ion with the same sign of charge as that of the watery phase for the reason that this ion diminishes the charge on the solution side of the membrane. When, therefore, the ions of an electrolyte raise the charge on the unit area of the membrane on the solution side above that on the side of pure water, a flow of the oppositely charged liquid must occur through the interstices of the membrane from the side of the water to the side of the solution (positive osmosis). When, however, the ions of an electrolyte lower the charge on the unit area of the solution side of the membrane below that on the pure water side of the membrane, liquid will diffuse from the solution into the pure water (negative osmosis). 4. We must, furthermore, conclude that in lower concentrations of many electrolytes the density of electrification of the double layer increases with an increase in concentration, while in higher concentrations of the same electrolytes it decreases with an increase in concentration. The turning point lies for a number of electrolytes at a molecular concentration of about M/512 or M/256. This explains why in lower concentrations of electrolytes the rate of diffusion of water through a collodion membrane from pure water into solution rises at first rapidly with an increase in concentration while beyond a certain concentration (which in a number of electrolytes is M/512 or M/256) the rate of diffusion of water diminishes with a further increase in concentration.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IX. WATER UPTAKE AND SWELLING OF COLLODION MEMBRANES IN AQUEOUS SOLUTIONS OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES

1. Dried collodion membranes are known to swell in water and to the same limited extent also in solutions of strong inorganic electrolytes (Carr and Sollner). The present investigation shows that in solutions of organic electrolytes and non-electrolytes, the swelling of dried collodion membranes is not as uniform, but depends on the nature of the solute. 2. The solutions of typically "hydrophilic" substances, e.g., glycerine, glucose, and citric acid, swell collodion membranes only to the same extent as water and solutions of strong electrolytes. In solutions of typically carbophilic substances (e.g., butyric acid, valeric acid, isobutyl alcohol, valeramide, phenol, and m-nitrophenol) the swelling of the membranes is much stronger than in water, according to the concentration used. For the brand of collodion used the swelling in 0.5 M solution was in some cases as high as 26 per cent of the original volume, as compared to 6 to 7 per cent in water. Therefore, in these solutions the "water-wetted dried" collodion membrane is not rigid, inert, and non-swelling, but behaves as a swelling membrane. 3. The solutes which cause an increased swelling of the membranes are accumulated in the latter, the degree of accumulation being markedly parallel with the degree of their specific swelling action. 4. The anomalously high permeabilities of certain carbophilic organic solutes reported by Michaelis, Collander, and Höber find an explanation in the specific interaction of these substances with collodion. 5. The use of the collodion membrane as a model of the ideal porous membrane is restricted to those instances in which no specific interaction occurs between the solute and the collodion.     

INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON THE ELECTRIFICATION AND THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES

1. When a watery solution is separated from pure water by a collodion membrane, the initial rate of diffusion of water into the solution is influenced in an entirely different way by solutions of electrolytes and of non-electrolytes. Solutions of non-electrolytes, e.g. sugars, influence the initial rate of diffusion of water through the membrane approximately in direct proportion to their concentration, and this. influence begins to show itself under the conditions of our experiments when the concentration of the sugar solution is above M/64 or M/32. We call this effect of the concentration of the solute on the initial rate of diffusion of water into the solution the gas pressure effect. 2. Solutions of electrolytes show the gas pressure effect upon the initial rate of diffusion also, but it commences at a somewhat higher concentration than M/64; namely, at M/16 or more (according to the nature of the electrolyte). 3. Solutions of electrolytes of a lower concentration than M/16 or M/8 have a specific influence on the initial rate of diffusion of water through a collodion membrane from pure solvent into solution which is not found in the case of the solutions of non-electrolytes and which is due to the fact that the particles of water diffuse in this case through the membrane in an electrified condition, the sign of the charge depending upon the nature of the electrolyte in solution, according to two rules given in a preceding paper. 4. In these lower concentrations the curves representing the influence of the concentration of the electrolyte on the initial rate of diffusion of water into the solution rise at first steeply with an increase in the concentration, until a maximum is reached at a concentration of M/256 or above. A further increase in concentration causes a drop-in the curve and this drop increases with a further increase of concentration until that concentration of the solute is reached in which the gas pressure effect begins to prevail; i.e., above M/16. Within a range of concentrations between M/256 and M/16 or more (according to the nature of the electrolyte) we notice the reverse of what we should expect on the basis of van''t Hoff''s law; namely, that the attraction of a solution of an electrolyte for water diminishes with an increase in concentration. 5. We wish to make no definite assumption concerning the origin of the electrification of water and concerning the mechanism whereby ions influence the rate of diffusion of water particles through collodion membranes from pure solvent to solution. It will facilitate, however, the presentation of our results if it be permitted to present them in terms of attraction and repulsion of the charged particles of water by the ions. With this reservation we may say that in the lowest concentrations attraction of the electrified water particles by the ions with the opposite charge prevails over the repulsion of the electrified water particles by the ions with the same sign of charge as that of the water; while beyond a certain critical concentration the repelling action of the ion with the same sign of charge as that of the water particles upon the latter increases more rapidly with increasing concentration of the solute than the attractive action of the ion with the opposite charge. 6. It is shown that negative osmosis, i.e. the diminution of the volume of the solution of acids and of alkalies when separated by collodion membranes from pure water, occurs in the same range of concentrations in which the drop in the curves of neutral salts occurs, and that it is due to the same cause; namely, the repulsion of the electrified particles of water by the ion with the same sign of charge as that of the water. This conclusion is supported by the fact that negative osmosis becomes pronounced when the ion with the same sign of charge as that of the electrified particles of water carries more than one charge.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

THE COLLOIDAL BEHAVIOR OF PROTEINS

1. It is well known that neutral salts depress the osmotic pressure, swelling, and viscosity of protein-acid salts. Measurements of the P.D. between gelatin chloride solutions contained in a collodion bag and an outside aqueous solution show that the salt depresses the P.D. in the same proportion as it depresses the osmotic pressure of the gelatin chloride solution. 2. Measurements of the hydrogen ion concentration inside the gelatin chloride solution and in the outside aqueous solution show that the difference in pH of the two solutions allows us to calculate the P.D. quantitatively on the basis of the Nernst formula See PDF for Equation if we assume that the P.D. is due to a difference in the hydrogen ion concentration on the two sides of the membrane. 3. This difference in pH inside minus pH outside solution seems to be the consequence of the Donnan membrane equilibrium, which only supposes that one of the ions in solution cannot diffuse through the membrane. It is immaterial for this equilibrium whether the non-diffusible ion is a crystalloid or a colloid. 4. When acid is added to isoelectric gelatin the osmotic pressure rises at first with increasing hydrogen ion concentration, reaches a maximum at pH 3.5, and then falls again with further fall of the pH. It is shown that the P.D. of the gelatin chloride solution shows the same variation with the pH (except that it reaches its maximum at pH of about 3.9) and that the P.D. can be calculated from the difference of pH inside minus pH outside on the basis of Nernst''s formula. 5. It was found in preceding papers that the osmotic pressure of gelatin sulfate solutions is only about one-half of that of gelatin chloride or gelatin phosphate solutions of the same pH and the same concentration of originally isoelectric gelatin; and that the osmotic pressure of gelatin oxalate solutions is almost but not quite the same as that of the gelatin chloride solutions of the same pH and concentration of originally isoelectric gelatin. It was found that the curves for the values for P.D. of these four gelatin salts are parallel to the curves of their osmotic pressure and that the values for pH inside minus pH outside multiplied by 58 give approximately the millivolts of these P.D. In this preliminary note only the influence of the concentration of the hydrogen ions on the P.D. has been taken into consideration. In the fuller paper, which is to follow, the possible influence of the concentration of the anions on this quantity will have to be discussed.     

ELECTROPHORESIS OF BACTERIA AS INFLUENCED BY HYDROGEN ION CONCENTRATION AND THE PRESENCE OF SODIUM AND CALCIUM SALTS

1. We have confirmed the results of earlier workers particularly of Northrop and De Kruif in regard to the following points: (a) the general tendency of the bacterial cell when suspended in distilled water near the zone of neutrality to move toward the anode of an electrical field; (b) the fact that the migration of bacterial cells in the electrical field is a function of the reaction of the menstruum. The curve obtained by plotting velocity of migration against pH passes through an isoelectric point at about pH 3.0, at greater acidity the direction of migration becomes reversed (toward the cathode) and in still more acid solution (pH = 1.0) again disappears; while at reactions less acid than pH 3.0 the velocity is toward the anode and increases with increasing alkalinity; (c) the fact that neutral salts depress the velocity of migration, calcium salts being much more effective than sodium salts of the same concentration. 2. We further find: (a) that on the extreme alkaline side of the curve of velocity of migration plotted against pH a maximum value is reached at about pH 10 with a fall at about pH 12.0 which in many experiments reaches an isopotential point; (b) that the depressing effect of salts is accompanied by a general shifting of the curve of migration velocity so that a maximum velocity (of course absolutely less than that manifest in the absence of salts) appears at about pH 7.0 and an abolition of velocity at pH 9.0 to 10.0; (c) that an apparent "antagonistic" effect is indicated between CaCl₂ and NaCl, the presence of a certain concentration of the latter salt diminishing to a slight but definite degree the depressing effect produced by the former; (d) that heat-killed bacterial cells exhibit essentially the same curve of migration velocity as that of the living cells; (e) that bacterial spores exhibit the same general curve of migration velocity as vegetative cells, although the actual velocity is apparently slightly less. 3. All of the observed phenomena appear to be in accord with the assumption that marked differences in dielectric constants did not appear under the conditions studied and if this assumption be granted the results are in accord with the fundamental postulates of the Donnan equilibrium as applied to the explanation of the origin of potential difference between a bacterial cell and its enveloping menstruum. It is possible but not at all certain that the phenomenon of antagonism may require the introduction of additional assumptions for its explanation. Professor Donnan and other investigators have clearly understood the importance of applying the concept of membrane equilibria in the elucidation of physiological phenomena. Our findings add to the numerous vindications favoring this view and emphasize the importance of further study of membrane equilibria in bacterial suspensions. We have pointed out that certain potential differences between bacteria and their menstrua are apparently associated with some of the phenomena of viability. Viability and potential differences may, however, under certain conditions vary quite independently as evidenced by the fact that normal rates of migration are demonstrable after the cells have been killed by heat. Thus, considerable caution must be exercised in relating the existence of these charges to the metabolism of the cell.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : I. MEMBRANE POTENTIALS.

1. It is shown that a neutral salt depresses the potential difference which exists at the point of equilibrium between a gelatin chloride solution contained in a collodion bag and an outside aqueous solution (without gelatin). The depressing effect of a neutral salt on the P.D. is similar to the depression of the osmotic pressure of the gelatin chloride solution by the same salt. 2. It is shown that this depression of the P.D. by the salt can be calculated with a fair degree of accuracy on the basis of Nernst''s logarithmic formula on the assumption that the P.D. which exists at the point of equilibrium is due to the difference of the hydrogen ion concentration on the opposite sides of the membrane. 3. Since this difference of hydrogen ion concentration on both sides of the membrane is due to Donnan''s membrane equilibrium this latter equilibrium must be the cause of the P.D. 4. A definite P.D. exists also between a solid block of gelatin chloride and the surrounding aqueous solution at the point of equilibrium and this P.D. is depressed in a similar way as the swelling of the gelatin chloride by the addition of neutral salts. It is shown that the P.D. can be calculated from the difference in the hydrogen ion concentration inside and outside the block of gelatin at equilibrium. 5. The influence of the hydrogen ion concentration on the P.D. of a gelatin chloride solution is similar to that of the hydrogen ion concentration on the osmotic pressure, swelling, and viscosity of gelatin solutions, and the same is true for the influence of the valency of the anion with which the gelatin is in combination. It is shown that in all these cases the P.D. which exists at equilibrium can be calculated with a fair degree of accuracy from the difference of the pH inside and outside the gelatin solution on the basis of Nernst''s logarithmic formula by assuming that the difference in the concentration of hydrogen ions on both sides of the membrane determines the P.D. 6. The P.D. which exists at the boundary of a gelatin chloride solution and water at the point of equilibrium can also be calculated with a fair degree of accuracy by Nernst''s logarithmic formula from the value pCl outside minus pCl inside. This proves that the equation x² = y ( y + z) is the correct expression for the Donnan membrane equilibrium when solutions of protein-acid salts with monovalent anion are separated by a collodion membrane from water. In this equation x is the concentration of the H ion (and the monovalent anion) in the water, y the concentration of the H ion and the monovalent anion of the free acid in the gelatin solution, and z the concentration of the anion in combination with the protein. 7. The similarity between the variation of P.D. and the variation of the osmotic pressure, swelling, and viscosity of gelatin, and the fact that the Donnan equilibrium determines the variation in P.D. raise the question whether or not the variations of the osmotic pressure, swelling, and viscosity are also determined by the Donnan equilibrium.     

THE INFLUENCE OF ELECTROLYTES ON THE CATAPHORETIC CHARGE OF COLLOIDAL PARTICLES AND THE STABILITY OF THEIR SUSPENSIONS : II. EXPERIMENTS WITH PARTICLES OF GELATIN,CASEIN, AND DENATURED EGG ALBUMIN.

1. This paper gives measurements of the influence of various electrolytes on the cataphoretic P.D. of particles of collodion coated with gelatin, of particles of casein, and of particles of boiled egg albumin in water at different pH. The influence of the same electrolyte was about the same in all three proteins. 2. It was found that the salts can be divided into two groups according to their effect on the P.D. at the isoelectric point. The salts of the first group including salts of the type of NaCl, CaCl₂, and Na₂SO₄ affect the P.D. of proteins at the isoelectric point but little; the second group includes salts with a trivalent or tetravalent ion such as LaCl₃ or Na₄Fe(CN)₆. These latter salts produce a high P.D. on the isoelectric particles, LaCl₃ making them positively and Na₄Fe(CN)₆ making them negatively charged. This difference in the action of the two groups of salts agrees with the observations on the effect of the same salts on the anomalous osmosis through collodion membranes coated with gelatin. 3. At pH 4.0 the three proteins have a positive cataphoretic charge which is increased by LaCl₃ but not by NaCl or CaCl₂, and which is reversed by Na₄Fe(CN)₆, the latter salt making the cataphoretic charge of the particles strongly negative. 4. At pH 5.8 the protein particles have a negative cataphoretic charge which is strongly increased by Na₄Fe(CN)₆ but practically not at all by Na₂SO₄ or NaCl, and which is reversed by LaCl₃. the latter salt making the cataphoretic charge of the particles strongly positive. 5. The fact that electrolytes affect the cataphoretic P.D. of protein particles in the same way, no matter whether the protein is denatured egg albumin or a genuine protein like gelatin, furnishes proof that the solutions of genuine proteins such as crystalline egg albumin or gelatin are not diaphasic systems, since we shall show in a subsequent paper that proteins insoluble in water, e.g. denatured egg albumin, are precipitated when the cataphoretic P.D. falls below a certain critical value, while water-soluble proteins, e.g. genuine crystalline egg albumin or gelatin, stay in solution even if the P.D. of the particles falls below the critical P.D.     

THE ISOLATION OF A CELL MEMBRANE FRACTION FROM RAT LIVER

A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO₃, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ~0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.     

COUNTERACTION OF THE INHIBITING EFFECTS OF VARIOUS SUBSTANCES ON NITELLA

When living cells of Nitella are first exposed to (1) phosphate buffer mixture, or (2) phosphoric acid, or (3) hydrochloric acid, or (4) sodium chloride, or (5) sodium borate, and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, the rate of penetration of the dye into the vacuole is decreased as compared with the rate in the case of cells transferred directly from tap water to the same dye solution. When cells exposed to any one of these solutions are placed in the dye solution made up with phosphate buffer solution at pH 7.85, the rate of penetration of dye into the vacuole is the same as the rate in the case of cells transferred from the tap water to the same dye solution. It is probable that this removal of the inhibiting effect is due primarily to the presence of certain concentration of sodium and potassium ions in the phosphate buffer solution. If a sufficient concentration of sodium ions is added to the dye made up with a borate buffer mixture the inhibiting effect is removed just as it is in the case of the dye made up with the phosphate buffer mixture. The inhibiting effect of some of these substances is found to be removed by the dye containing a sufficient concentration of bivalent cations, or by washing the cells with salts of bivalent cations. The inhibiting effect and its removal are discussed from a theoretical standpoint.     

STUDIES ON SALT ACTION : X. THE INFLUENCE OF ELECTROLYTES UPON THE VIABILITY AND ELECTROPHORETIC MIGRATION OF BACTERIUM COLI.

1. The strain of Bacterium coli used in these experiments multiplies in distilled water at pH 6.0 and pH 8.0 and in Ringer-Locke solution at pH 6.0. Under all the other conditions studied the numbers decrease with the passage of time. 2. The electrophoretic charge of the cells is highest in distilled water at pH 6.0 and pH 8.0. Under all other conditions studied the velocity of migration is decreased, but the decrease is immediate and is not affected by more prolonged exposure. 3. A strongly acid solution (pH 2.0) causes a rapid death of the cells and a sharp decrease in electrophoretic charge, sometimes leading to complete reversal. 4. A strongly alkaline solution (pH 11.0) is almost as toxic as a strongly acid one, although in distilled water the organisms survive fairly well at this reaction. Electrophoretic charge, on the other hand, is only slightly reduced in such an alkaline medium. 5. In distilled water, reactions near the neutral point are about equally favorable to both viability and electrophoretic charge, pH 8.0 showing slightly greater multiplication and a slightly higher charge than pH 11.0. In the presence of salts, however, pH 8.0 is much less favorable to viability and somewhat more favorable to electrophoretic charge than is pH 6.0. 6. Sodium chloride solutions, in the concentrations studied, all proved somewhat toxic and all tended to depress electrophoretic charge. Very marked toxicity was, however, exhibited only in a concentration of .725 M strength or over and at pH 8.0, while electrophoretic migration velocity was only slightly decreased at a concentration of .0145 M strength. 7. Calcium chloride was more toxic than NaCl, showing very marked effects in .145 M strength at pH 8.0 and in 1.45 M strength at pH 6.0. It greatly depressed electrophoretic charge even in .0145 M concentration. 8. Ringer-Locke solution proved markedly stimulating to the growth of the bacteria at pH 6.0 while at pH 8.0 it was somewhat toxic, though less so than the solutions of pure salts. It depressed migration velocity at all pH values, being more effective than NaCl in this respect, but less effective than CaCl₂. 9. It would appear from these experiments that a balanced salt solution (Ringer-Locke''s) may be distinctly favorable to bacterial viability in water at an optimum reaction while distinctly unfavorable in a slightly more alkaline solution. 10. Finally, while there is a certain parallelism between the influence of electrolytes upon viability and upon electrophoretic charge, the parallelism is not a close one and the two effects seem on the whole to follow entirely different laws.