STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : IV. PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA FLEXILIS

Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance. Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue. When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

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.     

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.     

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.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

EXIT OF DYE FROM LIVING CELLS OF NITELLA AT DIFFERENT pH VALUES

Experiments on the exit of brilliant cresyl blue from the living cells of Nitella, in solutions of varying external pH values containing no dye, confirm the theory that the relation of the dye in the sap to that in the external solution depends on the fact that the dye exists in two forms, one of which (DB) can pass through the protoplasm while the other (DS) passes only slightly. DB increases (by transformation of DS to DB) with an increase in the pH value, and is soluble in substances like chloroform and benzene. DS increases with decrease in pH value and is insoluble (or nearly so) in chloroform and benzene. The rate of exit of the dye increases as the external pH value decreases. This may be explained on the ground that DB as it comes out of the cell is partly changed to DS, the amount transformed increasing as the pH value decreases. The rate of exit of the dye is increased when the pH value of the sap is increased by penetration of NH₃.     

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.     

AUTORADIOGRAPHIC ANALYSIS ON AGAR PLATES OF ANTIGENS FROM SUB CELLULAR FRACTIONS OF RAT LIVER SLICES

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of ¹⁴C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.     

ACCUMULATION OF BRILLIANT CRESYL BLUE IN THE SAP OF LIVING CELLS OF NITELLA IN THE PRESENCE OF NH3

When the living cells of Nitella are placed in a solution of brilliant cresyl blue containing NH₄Cl, the rate of accumulation of the dye in the sap is found to be lower than when the cells are placed in a solution of dye containing no NH₄Cl and this may occur without any increase in the pH value of the cell sap. This decrease is found to be primarily due to the presence of NH₃ in the sap and seems not to exist where NH₃ is present only in the external solution at the concentration used.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

ON THE ACCUMULATION OF DYE IN NITELLA

Living cells of Nitella were placed in different concentrations of brilliant cresyl blue solutions at pH 6.9. It was found that the greater the concentration of the external dye solution, the greater was the speed of accumulation of the dye in the cell sap and higher was the concentration of dye found in the sap at equilibrium. Analysis of the time curves showed that the process may be regarded as a reversible pseudounimolecular reaction. When the concentration in the sap is plotted as ordinates and the concentration in the outside solution as abscissae the curve is convex toward the abscissae. There is reason to believe that secondary changes involving injury take place as the dye accumulates and that if these changes did not occur the curve would be concave toward the abscissae. The process may be explained as a chemical combination of the dye with a constituent of the cell. This harmonizes with the fact that the temperature coefficient is high (about 4.9). Various other possible explanations are discussed.     

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 INFLUENCE OF LIGHT,TEMPERATURE, AND OTHER CONDITIONS ON THE ABILITY OF NITELLA CELLS TO CONCENTRATE HALOGENS IN THE CELL SAP

1. By the use of a special analytical technique it has been possible to study the accumulation of halogens in the cell sap of Nitella. 2. From a dilute solution, Br may be accumulated in the sap in a concentration much greater than that of the external solution. The conductivity of the sap may be markedly increased by such accumulation. The process is a slow one so that a month or more may be required to approach equilibrium. 3. Cl may be lost from the cell as a result of the accumulation of Br and vice versa. Other reciprocal relations between Cl and Br are indicated. 4. At equilibrium practically as much Br accumulated in the sap with an external solution containing 1 milli-equivalent of Br as with one containing 5 milli-equivalents. 5. Light energy was indispensable to the accumulation of Br. The temperature coefficient was characteristic of a chemical process.     

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 EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

THE PENETRATION OF DYES AS INFLUENCED BY HYDROGEN ION CONCENTRATION

When cells of Nitella are placed in buffer solutions at pH 9, there is a very slow and gradual increase in the pH of the sap from pH 5.6 to 6.4 (when death of the cells takes place). If the living cells are placed in 0.002 per cent dye solutions of brilliant cresyl blue at different pH values (from pH 6.6 to pH 9), it is found that the rate of penetration of the dye, and the final equilibrium attained, increases with increase in pH value, which can be attributed to an increase in the active protein (or other amphoteric electrolyte) in the cell which can combine with the dye.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA

The protoplasm of Valonia macrophysa forms a delicate layer, only a few microns in thickness, which contains numerous chloroplasts and nuclei. The outer surface is in contact with the cell wall, the inner with the vacuolar sap. As far as microscopic observation goes, these two surfaces seem alike; but measurements of potential difference indicate that they are decidedly different. We find that the chain sap | protoplasm | sap gives about 14.5 millivolts, the inner surface being positive to the outer. In order to explain this we may assume that the protoplasm consists of layers, the outer surface, X, differing from the inner surface, Y, and from the body of the protoplasm, W. We should then have the unsymmetrical chain sap | X | W | Y | sap which could produce an electromotive force. If the two surfaces of such a very thin layer of protoplasm can be different, it is of fundamental significance for the theory of the nature of living matter.     

Variation in the components of palm wine during fermentation

Palm sap from Raphia vinifera was autofermented to palm wine. The pH of the sap fell from 7.0 to 4.5 in 120 h. During this autofermentation the components of the palm sap varied. Quantitative analyses indicated that fresh unfermented sap contained 3.92% (w/v) glucose, 10 mg/ml Vitamin C, 4 mg/ml protein and 0% (w/v) alcohol.     

THE BEHAVIOR OF CHLORIDES IN THE CELL SAP OF NITELLA

1. A method is given for determining the chloride content in a drop (less than 0.03 cc.) of the cell sap of Nitella. 2. Chlorides accumulate in the sap to the extent of 0.128 M; this accumulation can be followed during the growth of the cell. The chloride content does not increase when the cell is placed for 2 days in solutions (at pH 6.2) containing chlorides up to 0.128 M. 3. The exosmosis of chlorides from injured cells can be followed quantitatively. When one end of the cell is cut off a wave of injury progresses toward the other end; this is accompanied by a progressive exosmosis of chlorides.