THE KINETICS OF PENETRATION : XIX. ENTRANCE OF ELECTROLYTES AND OF WATER INTO IMPALED HALICYSTIS

When cells of Halicystis are impaled on a capillary so that space is provided into which the sap can migrate, the rate of entrance of water and of electrolyte is increased about 10-fold. In impaled Valonia cells the rate is increased about 15-fold. After a relatively rapid non-linear rate of increase of sap volume immediately after impalement (which may possibly represent the partial dissipation of the difference of the osmotic energy between intact and impaled cells) the volume increases at a linear rate, apparently indefinitely. Since the halide concentration of the sap at the end of the experiment is (within the limits of natural variation) the same as in the intact cell, we conclude that electrolyte also enters the sap about 10 times as fast as in the intact cell. As in the case of Valonia we conclude that there is a mechanism whereby in the intact cell the osmotic concentration of the sap is prevented from greatly exceeding that of the sea water. This may be associated with the state of hydration of the non-aqueous protoplasmic surfaces.     

THE KINETICS OF PENETRATION : III. EQUATIONS FOR THE EXCHANGE OF IONS

Equations are given for a variety of cases when ions of the same sign are exchanged between the interior of a living cell and the external solution.     

THE KINETICS OF PENETRATION : XVIII. ENTRANCE OF WATER INTO IMPALED HALICYSTIS

The rate of entrance of water into impaled cells of Halicystis Osterhoutii, Blinks and Blinks, has been determined directly by measurements of the rise of sap in a capillary for dilute sea waters (containing between 90 and 30 per cent sea water). The velocity constant remains reasonably constant down to 50 per cent sea water but it decreases markedly in lower concentrations.     

THE ACCUMULATION OF ELECTROLYTES : I. THE ENTRANCE OF AMMONIA INTO VALONIA MACROPHYSA

When 0.005 M NH₄Cl is added to sea water containing cells of Valonia macrophysa ammonia soon appears in the sap and may reach a concentration inside over 40 times as great as outside. It appears to enter as undissociated NH₃ (or NH₄OH) and tends to reach a pseudoequilibrium in which the activity of undissociated NH₃ (or NH₄OH) is the same inside and outside. When ammonia first enters, the pH value of the sap rapidly rises but it soon reaches a maximum and subsequently falls off. At the same time there is an increase of halide in the sap which, however, does not run a parallel course to the ammonia accumulation, but it comes to a new equilibrium value and remains constant. The increase in NH₃ in the sap is accompanied by a decrease in the concentration of K. As NH₃ enters the specific gravity of the sap decreases and the cells rise to the surface and continue to grow as floating organisms. The growth of the cells is increased.     

THE PENETRATION OF STRONG ELECTROLYTES

The entrance of strong electrolytes into Valonia is very slow unless the cells are injured. This, together with the very high electrical resistance of the protoplasm, suggests that they may penetrate largely as undissociated molecules formed at the surface of the protoplasm by the collision of ions. Under favorable circumstances KCl may be absorbed to the extent of 3 x 10^–8 mols per hour per sq. cm. of surface together with about 0.17 as much NaCl. Other substances which seem to penetrate to some extent are Li, Rb, Br, BrO₃, I, IO₃, and selenite. Little or no penetration is shown by SCN, ferricyanide, ferrocyanide, formate, salicylate, tungstate, seleniate, NO₂, SO₃, Sb, glycerophosphate, and many heavy metals and the alkaline earths. In sea water whose specific gravity had been increased by CsCl cells of Valonia floated for over a year and there was little or no penetration of Cs except as the result of injury. The penetration of NH₄Cl decreases the specific gravity of the sap and causes the cells to float: under these circumstances they live indefinitely. It is probable that NH₃ or NH₄OH penetrates and is subsequently changed to NH₄Cl. It would seem that if the sea contained a little more ammonia this would be a floating organism.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.     

THE KINETICS OF PENETRATION : VI. SOME FACTORS AFFECTING PENETRATION

Some of the factors affecting penetration in living cells may be advantageously studied in models in which the organic salts KG and NaG diffuse from an aqueous solution A, through a non-aqueous layer B (representing the protoplasmic surface) into an aqueous solution C (representing the sap and hence called artificial sap) where they react with CO₂ to form KHCO₃ and NaHCO₃. Their relative proportions in C depend chiefly on the partition coefficients and on the diffusion constants in the non-aqueous layer. But the ratio is also affected by other variables, among which are the following: 1. Temperature, affecting diffusion constants and partition coefficients and altering the thickness of the unstirred layers by changing viscosity. 2. Viscosity (especially in the non-aqueous layers) which depends on temperature and the presence of solutes. 3. Rate of stirring, which affects the thickness of the unstirred layers and the transport of electrolyte in those that are stirred. 4. Shape and surface area of the non-aqueous layer. 5. Surface forces. 6. Reactions occurring at the outer surface such as loss of water by the electrolyte or its molecular association in the non-aqueous phase. The reverse processes will occur at the inner surface and here also combinations with acids or other substances in the "artificial sap" may occur. 7. Outward diffusion from the artificial sap. The outward movement of KHCO₃ and NaHCO₃ is small compared with the inward movement of KG and NaG when the concentrations are equal. This is because the partition coefficients³ of the bicarbonates are very low as compared with those of NaG and KG. Since CO₂ and HCO₃ ^- diffuse into A and combine with KG and NaG the inward movement of potassium and sodium falls off in proportion as the concentration of KG and NaG is lessened. 8. Movement of water into the non-aqueous phase and into the artificial sap. This may have a higher temperature coefficient than the penetration of electrolytes. 9. Variation of the partition coefficients with concentration and pH. Many of these variables may occur in living cells. (It happens that the range of variation in the ratio of potassium to sodium in the models resembles that found in Valonia.)     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

THE KINETICS OF PENETRATION : VIII. TEMPORARY ACCUMULATION

A model is described which throws light on the mechanism of accumulation. In the model used an external aqueous phase A is separated by a non-aqueous phase B (representing the protoplasm) from the artificial sap in C. A contains KOH and C contains HCl: they tend to mix by passing through the non-aqueous layer but much more KOH moves so that most of the KCl is formed in C, where the concentration of potassium becomes much greater than in A. This accumulation is only temporary for as the system approaches equilibrium the composition of A approaches identity with that of C, since all the substances present can pass through the non-aqueous layer. Such an approach to equilibrium may be compared to the death of the cell as the result of which accumulation disappears. During the earlier stages of the experiment potassium tends to go in as KOH and at the same time to go out as KCl. These opposing tendencies do not balance until the concentration of potassium inside becomes much greater than outside (hence potassium accumulates). The reason is that KCl, although its driving force be great, moves very slowly in B because its partition coefficient is low and in consequence its concentration gradient in B is small. This illustrates the importance of partition coefficients for penetration in models and in living cells. It also indicates that accumulation depends on the fact that permeability is greater for the ingoing compound of the accumulating substance than for the outgoing compound. Other things being equal, accumulation is increased by maintaining a low pH in C. Hence we may infer that anything which checks the production of acid in the living cell may be expected to check accumulation and growth. This model recalls the situation in Valonia and in most living cells where potassium accumulates as KCl, perhaps because it enters as KOH and forms KA in the sap (where A is an organic anion). In some plants potassium accumulates as KA but when HCl exists in the external solution it will tend to enter and displace the weaker acid HA (if this be carbonic it can readily escape): hence potassium may accumulate to a greater or less extent as KCl. Injury of the cell may produce a twofold effect, (1) increase of permeability, (2) lessened accumulation. The total amount of electrolyte taken up in a given time will be influenced by these factors and may be greater than normal in the injured cell or less, depending somewhat on the length of the interval of time chosen.     

THE KINETICS OF PENETRATION : XV. THE RESTRICTION OF THE CELLULOSE WALL

When Valonia cells are impaled on capillaries, it is in some ways equivalent to removing the comparatively inelastic cellulose wall. Under these conditions sap can migrate into a free space and it is found that on the average the rate of increase of volume of the sap is 15 times what it is in intact cells kept under comparable conditions. The rate of increase of volume is a little faster during the first few hours of the experiment, but it soon becomes approximately linear and remains so as long as the experiment is continued. The slightly faster rate at first may mean that the osmotic pressure of the sap is approaching that of the sea water (in the intact cell the sap osmotic pressure is always slightly above that of the sea water). This might result from a more rapid entrance of water than of electrolyte, as would be expected when the restriction of the cellulose wall was removed. During the linear part of the curve the osmotic concentration and the composition of the sap suffer no change, so that entrance of electrolyte must be 15 times as fast in the impaled cells as it is in the intact cells. The explanation which best accords with the facts is that in the intact cell the entrance of electrolyte tends to increase the osmotic pressure. As a consequence the protoplasm is partially dehydrated temporarily and it cannot take up more water until the cellulose wall grows so that it can enclose more volume. The dehydration of the protoplasm may have the effect of making the non-aqueous protoplasm less permeable to electrolytes by reducing the diffusion and partition coefficients on which the rate of entrance depends. In this way the cell is protected against great fluctuations in the osmotic concentration of the sap.     

THE KINETICS OF PENETRATION : IX. MODELS OF MATURE CELLS

To imitate cells which have ceased to grow we have made models in which artificial sap is separated from the external solution by a non-aqueous layer (representing the protoplasm). A stream of CO₂ is bubbled through the artificial sap to imitate its production by the living cell. Potassium passes from the external solution through the non-aqueous layer into the artificial sap and there reacts with CO₂ to form KHCO₃: its rate of entrance depends on the supply of CO₂. Hence the increase of volume depends on the supply of CO₂ (as is probably true of the living cell). By regulating the supply of CO₂ and the osmotic pressure we are able to keep the volume and composition of the artificial sap approximately constant while maintaining a higher concentration of potassium than in the external solution. In these respects the model resembles certain mature cells which have ceased to grow.     

THE KINETICS OF PENETRATION : V. THE KINETICS OF A MODEL AS RELATED TO THE STEADY STATE

An organic potassium salt, KG, passes from an aqueous phase, A, through a non-aqueous layer, B, into a watery solution, C. In C it reacts with CO₂ to form KHCO₃. The ionic activity product (K) (G) in C is thus kept at such a low level that KG continues to diffuse into C after the concentration of potassium becomes greater in C than in A. Hence potassium accumulates in C, the osmotic pressure rises, and water goes in. A steady state is eventually reached in which potassium and water enter C in a constant ratio. The rate of entrance of potassium (with no water penetrating into C) may fall off in a manner approximately exponential. But water enters and may produce an exponential decrease in concentration. This suggests that the kinetics may be treated like that of two consecutive monomolecular reactions. Calculations made on this basis agree very well with the observed values. The rate of penetration appears to be proportional to the concentration gradient of KG in the non-aqueous layer and in consequence depends upon the partition coefficients which determine this gradient. Exchange of ions (passing as such through the non-aqueous layer) does not seem to play an important rôle in the entrance of potassium. The kinetics of the model may be similar to that of living cells.     

THE KINETICS OF PENETRATION : XVII. THE EXIT OF AMMONIA IN LIGHT AND DARKNESS

The exit of accumulated ammonia from the sap of Valonia macrophysa, Kütz., into normal (nearly ammonia-free) sea water, has been studied in light (alternation of daylight and darkness) and in darkness. Exit is always preceded by an induction period lasting 1 or more days. This is longer in darkness. After exit starts the rate is greater in light than in darkness. The pH of the sap drops off soon after the cells are exposed to normal sea water even before any definite decrease in the ammonia concentration of the sap has occurred. This suggests that the decrease in the pH is due to the loss of a very small amount of NH₃ or NH4OH without a corresponding gain of sodium as a base. In most cases sodium replaced the ammonia lost during exit, but there is some evidence that potassium may also replace ammonia. To account for the induction period it is suggested that other species than NH₄ X are concerned in the transport of ammonia, for example urea or amino acids.     

THE KINETICS OF PENETRATION : XVI. THE ACCUMULATION OF AMMONIA IN LIGHT AND DARKNESS

The accumulation of ammonia takes place more rapidly in light than in darkness. The accumulation appears to go on until a steady state is attained. The steady state concentration of ammonia in the sap is about twice as great in light as in darkness. Both effects are possibly due to the fact that the external pH (and hence the concentration of undissociated ammonia) outside is raised by photosynthesis. Certain "permeability constants" have been calculated. These indicate that the rate is proportional to the concentration gradient across the protoplasm of NH₄ X which is formed by the interaction of NH₃ or NH₄OH and HX, an acid elaborated in the protoplasm. The results are interpreted to mean that HX is produced only at the sap-protoplasm interface and that on the average its concentration there is about 7 times as great as at the sea water-protoplasm interface. This ratio of HX at the two surfaces also explains why the concentration of undissociated ammonia in the steady state is about 7 times as great in the sea water as in the sap. The permeability constant P'''''' appears to be greater in the dark. This is possibly associated with an increase in the concentration of HX at both interfaces, the ratio at the two surfaces, however, remaining about the same. The pH of sap has been determined by a new method which avoids the loss of gas (CO₂), an important source of error. The results indicate that the pH rises during accumulation but the extent of this rise is smaller than has hitherto been supposed. As in previous experiments, the entering ammonia displaced a practically equivalent amount of potassium from the sap and the sodium concentration remained fairly constant. It seems probable that the pH increase is due to the entrance of small amounts of NH₃ or NH₄OH in excess of the potassium lost as a base.     

KINETICS OF PENETRATION : VII. MOLECULAR VERSUS IONIC TRANSPORT

In some living cells the order of penetration of certain cations corresponds to that of their mobilities in water. This has led to the idea that electrolytes pass chiefly as ions through the protoplasmic surface in which the order of ionic mobilities is supposed to correspond to that found in water. If this correspondence could be demonstrated it would not prove that electrolytes pass chiefly as ions through the protoplasmic surface for such a correspondence could exist if the movement were mostly in molecular form. This is clearly shown in the models here described. In these the protoplasmic surface is represented by a non-aqueous layer interposed between two aqueous phases, one representing the external solution, the other the cell sap. The order of penetration through the non-aqueous layer is Cs > Rb > K > Na > Li. This will be recognized as the order of ionic mobilities in water. Nevertheless the movement is mostly in molecular form in the nonaqueous layer (which is used in the model to represent the protoplasmic surface) since the salts are very weak electrolytes in this layer. The chief reason for this order of penetration lies in the fact that the partition coefficients exhibit the same order, that of cesium being greatest and that of lithium smallest. The partition coefficients largely control the rate of entrance since they determine the concentration gradient in the non-aqueous layer which in turn controls the process of penetration. The relative molecular mobilities (diffusion constants) in the non-aqueous layer do not differ greatly. The ionic mobilities are not known (except for K⁺ and Na⁺) but they are of negligible importance, since the movement in the non-aqueous layer is largely in molecular form. They may follow the same order as in water, in accordance with Walden''s rule. Ammonium appears to enter faster than its partition coefficient would lead us to expect, which may be due to rapid penetration of NH₃. This recalls the apparent rapid penetration of ammonium in living cells which has also been explained as due to the rapid penetration of NH₃. Both observation and calculation indicate that the rate of penetration is not directly proportional to the partition coefficient but increases somewhat less rapidly. Many of these considerations doubtless apply to living cells.     

THE KINETICS OF PENETRATION : IV. DIFFUSION AGAINST A GROWING POTENTIAL GRADIENT IN MODELS

In a model consisting of a non-aqueous layer (representing the protoplasm) placed between an inner, more acid, aqueous layer (representing the sap) and an outer, more alkaline, aqueous solution (representing the external solution bathing a living cell) the penetration of potassium creates an outwardly directed potential against which potassium continues to diffuse inward, thereby increasing the outward potential. This continues until the steady state is reached. The potassium sets up less potential in entering than in escaping and the net result is an outwardly directed potential. A similar process appears to take place in certain living cells.     

THE ACCUMULATION OF ELECTROLYTES : VII. ORGANIC ELECTROLYTES PART 1

The inorganic constituents of the sap of Rheum (rhubarb), Rumex (field sorrel), and Oxalis (wood sorrel) show a great preponderance of cations over anions, as would be expected if the cations entered chiefly as hydrates (other possibilities will be discussed in Part 2).     

THE ACCUMULATION OF ELECTROLYTES : VII. ORGANIC ELECTROLYTES PART 2

Analyses have been made of the inorganic constituents of the juices expressed from the leaves of Rheum, Rumex, and Oxalis. It has been shown that in all cases there is a large excess of inorganic cations over anions in the sap, the average ratio of cations to anions being 3.8 (Part 1, p. 239). The ash analyses of plant tissues (chiefly leaves) reported in the literature have been examined critically, and it has been shown that the preponderance of inorganic cations over inorganic anions in the ash and in the sap is general. It has been concluded that the excess of inorganic cations is consistent with the view that cations pass into the protoplasm chiefly in the form of hydroxides, and are accumulated either in the form of organic salts (such as the oxalates) or in non-polar linkage. It has been concluded that practically all the potassium and sodium found in plant ash must have been present originally in the form of soluble ionogenic compounds, but that a considerable part of the calcium and magnesium may have been present originally in the form of insoluble salts or as components of non-polar compounds. The methods whereby the cations, particularly potassium, may have been accumulated have been discussed, and it has been concluded that as it does not seem very probable that they enter chiefly as nitrates or bicarbonates we may suppose that they go in to a large extent as hydrates: this is highly probable in the case which has been most carefully investigated (Valonia).     

THE KINETICS OF PENETRATION : XX. EFFECT OF pH AND OF LIGHT ON ABSORPTION IN IMPALED HALICYSTIS

The rate of entrance of electrolyte and of water into impaled cells of Halicystis Osterhoutii is unaffected by raising the pH of the sea water to 9.2 or lowering it to 7.0. It is quite possible that sodium enters by combining with an organic acid HX produced by the protoplasm. If the pK'' of this acid is sufficiently low the change in external pH would not produce much effect on the rate of entrance of sodium. The rate of entrance of electrolytes is affected by light. In normal light (i.e. natural succession of daylight and darkness) the rate is about twice as great as in darkness.     

CONTRIBUTION TO THE THEORY OF PERMEABILITY OF MEMBRANES FOR ELECTROLYTES

On page 39, Vol. viii, No. 2, September 18, 1925, multiply the right-hand side of formula (2) by the factor See PDF for Equation. On page 44, immediately after formula (1) the text should be continued as follows: Let us suppose a membrane to be separated by two solutions of KCl of different concentrations K₁ and K₂ and these concentrations and the corresponding concentrations of K⁺ within the membrane, which are in equilibrium with the outside solutions, to be so high that the H⁺ ions may be neglected. When a small electric current flows across the system, practically the K⁺ ions alone are transferred and that in a reversible manner. Therefore the total P.D. is practically See PDF for Equation This P.D. is composed of two P.D.''s at the boundaries and the diffusion potential within the membrane. Suppose the immobility of the anions is not absolute but only relative as compared with the mobility of the cations, KCl would gradually penetrate into the membrane to equal concentration with the outside solution on either side and no boundary potential would be established. In this case the diffusion P.D. within the membrane is the only P.D., amounting to See PDF for Equation but, V being practically = 0, it would result that See PDF for Equation So the definitive result is the same as in the former case. Now cancel the printed text as far as page 48, line 13 from the top of the page, but retain Fig. 1. On page 50, line 19 from the top of the page, cancel the sentence beginning with the word But and ending with the words of the chain.