CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : I. EFFECTS OF GUAIACOL ON VALONIA

In normal cells of Valonia the order of the apparent mobilities of the ions in the non-aqueous protoplasmic surface is K > Cl > Na. After treatment with 0.01 M guaiacol (which does not injure the cell) the order becomes Na > Cl > K. As it does not seem probable that such a reversal could occur with simple ions we may assume provisionally that in the protoplasmic surface we have to do with charged complexes of the type (KX _I)⁺, (KX _II)⁺, where X _I and X _II are elements or radicals, or with chemical compounds formed in the protoplasm. When 0.01 M guaiacol is added to sea water or to 0.6 M NaCl (both at pH 6.4, where the concentration of the guaiacol ion is negligible) the P.D. of the cell changes (after a short latent period) from about 10 mv. negative to about 28 mv. positive and then slowly returns approximately to its original value (Fig. 1, p. 14). This appears to depend chiefly on changes in the apparent mobilities of organic ions in the protoplasm. The protoplasmic surface is capable of so much change that it does not seem probable that it is a monomolecular layer. It does not behave like a collodion nor a protein film since the apparent mobility of Na⁺ can increase while that of K⁺ is decreasing under the influence of guaiacol.     

CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

COMPARATIVE STUDIES ON RESPIRATION : IX. THE EFFECTS OF ANTAGONISTIC SALTS ON THE RESPIRATION OF ASPERGILLUS NIGER.

1. In the presence of 0.05 per cent dextrose the respiration of Aspergillus niger is increased by NaCl in concentrations of 0.25 to 0.5M, and by 0.5M CaCl₂. 2. Stronger concentrations, as 2M NaCl and 1.25M CaCl₂, decrease the respiration. The decrease in the higher concentrations is probably an osmotic effect of these salts. 3. A mixture of 19 cc. of NaCl and 1 cc. of CaCl₂ (both 0.5M) showed antagonism, in that the respiration was normal, although each salt alone caused an increase. 4. Spores of Aspergillus niger did not germinate on 0.5M NaCl (plus 0.05 per cent dextrose) while they did on 0.5M CaCl₂ (plus 0.05 per cent dextrose) and on various mixtures of the two. This shows that a substance may have different effects on respiration from those which it has upon growth.     

CHEMICAL ANTAGONISM OF IONS : III. EFFECT OF SALT MIXTURES ON GELATIN ACTIVITY.

1.25 per cent gelatin solutions containing enough NaOH to bring them to pH 7.367 (or KOH to pH 7.203) were made up with various concentrations of NaCl, KCl and MgCl₂, alone and in mixtures, up to molar ionic strength. The effects of these salts on the pH were observed. MgCl₂ and NaCl alone lower the pH of the Na gelatinate or the K gelatinate, in all amounts of these salts. KCl first lowers the pH (up to 0.01 M K⁺), then raises the pH. Mixtures of NaCl and KCl (up to 0.09 M of the salt whose concentration is varied) raise the pH; then (up to 0.125 M Na⁺ or K⁺) lower the pH; and finally (above 0.125 M) behave like KCl alone. Mixtures of MgCl₂ and NaCl raise the pH up to 0.10 M Na⁺, and lower it up to 0.15 M Na⁺ regardless of the amount of MgCl ₂. Higher concentrations of NaCl have little effect, but the pH in this range of NaCl concentration is lowered with increase of MgCl₂. Mixtures of MgCl₂ and KCl behave as above described (for MgCl₂ and NaCl) and the addition of NaCl plus KCl to gelatin containing MgCl₂ produces essentially the same effect as the addition of either alone, except that the first two breaks in this curve come at 0.07 M and 0.08 M [Na⁺ + K⁺] and there is a third break at 0.12 M. In this pH range the free groups of the dicarboxylic acids and of lysine are essentially all ionized and the prearginine and histidine groups are essentially all non-ionized. The arginine group is about 84 per cent ionized. Hence we are studying a solution with two ionic species in equilibrium, one with the arginine group ionized, and one with it non-ionized. It is shown that the effect of each salt alone depends upon the effect of the cation on the activity of these two species due to combination. The anomalous effects of cation mixtures may be qualitatively accounted for if one or both of these species fail to combine with the cations in a mixture in proportion to the relative combination in solutions of each cation alone. Special precautions were taken to ensure accuracy in the pH measurements. The mother solutions gave identical readings to 0.001 pH and the readings with salts were discarded when not reproducible to 0.003 pH. All doubtful data were discarded.     

THE VARIATION OF ELECTRICAL RESISTANCE WITH APPLIED POTENTIAL : III. IMPALED VALONIA VENTRICOSA

Electrical resistance and polarization were measured during the passage of direct current across a single layer of protoplasm in the cells of Valonia ventricosa impaled upon capillaries. These were correlated with five stages of the P.D. existing naturally across the protoplasm, as follows: 1. A stage of shock after impalement, when the P.D. drops from 5 mv. to zero and then slowly recovers. There is very little effective resistance in the protoplasm, and polarization is slight. 2. The stage of recovery and normal P.D., with values from 8 to 25 mv. (inside positive). The average is 15 mv. At first there is little or no polarization when small potentials are applied in either direction across the protoplasm, nor when very large currents pass outward (from sap to sea water). But when the positive current passes inward there is a sudden response at a critical applied potential ranging from 0.5 to 2.0 volts. The resistance then apparently rises as much as 10,000 ohms in some cases, and the rise occurs more quickly in succeeding applications after the first. When the potential is removed there is a back E.M.F. displayed. Later there is also an effect of such inward currents which persists into the first succeeding outward flow, causing a brief polarization at the first application of the reverse potential. Still later this polarization occurs at every exposure, and at increasingly lower values of applied potentials. Finally there is a "constant" state reached in which the polarization occurs with currents of either direction, and the apparent resistance is nearly uniform over a considerable range of applied potential. 3. A state of increased P.D.; to 100 mv. (inside positive) in artificial sap; and to 35 or 40 mv. in dilute sea water or 0.6 M MgSO₄. The polarization response and apparent resistance are at first about as in sea water, but later decrease. 4. A reversed P.D., to 50 mv. (outside positive) produced by a variety of causes, especially by dilute sea water, and also by large flows of current in either direction. This stage is temporary and the cells promptly recover from it. While it persists the polarization appears to be much greater to outward currents than to inward. This can largely be ascribed to the reduction of the reversed P.D. 5. Disappearance of P.D. caused by death, and various toxic agents. The resistance and polarization of the protoplasm are negligible. The back E.M.F. of polarization is shown to account largely for the apparent resistance of the protoplasm. Its calculation from the observed resistance rises gives values up to 150 mv. in the early stages of recovery, and later values of 50 to 75 mv. in the "constant" state. These are compared with the back E.M.F. similarly calculated from the apparent resistance of intact cells. The electrical capacitance of the protoplasm is shown by the time curves to be of the order of 1 microfarad per cm.² of surface.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : II. THE ACTION OF GUAIACOL AS AFFECTED BY pH

The normal P.D. across the protoplasm of Valonia macrophysa is about 10 mv. negative (inwardly directed). On adding 0.01 M guaiacol to the sea water the P.D. becomes positive and then slowly returns approximately to the normal value. In many cases this behavior is not much affected by raising the pH and so increasing the concentration of the guaiacol ion but in other cases such an increase makes the P.D. somewhat more negative. But if we wait until the exposure to guaiacol has lasted 5 minutes (and the P.D. has returned to its normal value) before we raise the pH, the result is very different. The cell then behaves as though it had been sensitized to the action of the guaiacol ion which appears to be far more effective than undissociated guaiacol in making the P.D. more positive. This may be due in part to the high apparent mobility of the guaiacol ion and in part to alterations which it produces in the protoplasm (such alterations increase the P.D. across the protoplasm whereas ordinary injury would be expected to lower it and the cells live on after this treatment and show no signs of injury). This action of the guaiacol ion is in marked contrast to the behavior of other anions whose effect resembles that of Cl^-.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS

The cells of Halicystis impaled on capillaries reach a steady P.D. of 60 to 80 millivolts across the protoplasm from sap to sea water. The outer surface of the protoplasm is positive in the electrometer to the inner surface. The P.D. is reduced by contact with sap and balanced NaCl-CaCl₂ mixtures; it is abolished completely in solutions of NaCl, CaCl₂, KCl, MgSO₄, and MgCl₂. There is prompt recovery of P.D. in sea water after these exposures.     

MECHANICAL RESTORATION OF IRRITABILITY AND OF THE POTASSIUM EFFECT

Treatment of Nitella with distilled water apparently removes from the cell something which is responsible for the normal irritability and the potassium effect, (i.e. the large P.D. between a spot in contact with 0.01 M KCl and one in contact with 0.01 M NaCl). Presumably this substance (called R) is partially removed from the protoplasm by the distilled water. When this has happened a pinch which forces sap out into the protoplasm can restore its normal behavior. The treatment with distilled water which removes the potassium effect from the outer protoplasmic surface does not seem to affect the inner protoplasmic surface in the same way since the latter retains the outwardly directed potential which is apparently due to the potassium in the sap. But the inner surface appears to be affected in such fashion as to prevent the increase in its permeability which is necessary for the production of an action current. The pinch restores its normal behavior, presumably by forcing R from the sap into the protoplasm.     

DIFFERING RATES OF DEATH AT INNER AND OUTER SURFACES OF THE PROTOPLASM : I. EFFECTS OF FORMALDEHYDE ON NITELLA

When protoplasm dies it becomes completely and irreversibly permeable and this may be used as a criterion of death. On this basis we may say that when 0.2 M formaldehyde plus 0.001 M NaCl is applied to Nitella death arrives sooner at the inner protoplasmic surface than at the outer. If, however, we apply 0.17 M formaldehyde plus 0.01 M KCl death arrives sooner at the outer protoplasmic surface. The difference appears to be due largely to the conditions at the two surfaces. With 0.2 M formaldehyde plus 0.001 M NaCl the inner surface is subject to a greater electrical pressure than the outer and is in contact with a higher concentration of KCl. In the other case these conditions are more nearly equal so that the layer first reached by the reagent is the first to become permeable. The outer protoplasmic surface has the ability to distinguish electrically between K⁺ and Na⁺ (potassium effect). Under the influence of formaldehyde this ability is lost. This is chiefly due to a falling off in the partition coefficient of KCl in the outer protoplasmic surface. At about the same time the inner protoplasmic surface becomes completely permeable. But the outer protoplasmic surface retains its ability to distinguish electrically between different concentrations of the same salt, showing that it has not become completely permeable. After the potential has disappeared the turgidity (hydrostatic pressure inside the cell) persists for some time, probably because the outer protoplasmic surface has not become completely permeable.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

THE RELATIONS OF TEMPERATURE TO THE POTASSIUM EFFECT AND THE BIOELECTRIC POTENTIAL OF VALONIA

The effect of temperature upon the bioelectric potential across the protoplasm of impaled Valonia cells is described. Over the ordinary tolerated range, the P.D. is lowest around 25°C., rising both toward 15° and 35°. The time curves are characteristic also. The magnitude of the temperature effect can be controlled by changing the KCl content of the sea water (normally 0.012 M): the magnitude is greatly reduced at 0.006 M KCl, enhanced at 0.024 M, and greatly exaggerated at 0.1 M KCl. Conversely, temperature controls the magnitude of the potassium effect, which is smallest at 25°, with a cusped time course. It is increased, with a smoothly rising course, at 15°, and considerably enhanced, with only a small cusp, at 35°. A temporary "alteration" of the protoplasmic surface by the potassium is suggested to account for the time courses. This alteration does not occur at 15°; the protoplasm recovers only slowly and incompletely at 25°, but rapidly at 35°, in such fashion as to make the P.D. more negative than at 15°. This would account for the temperature effects observed in ordinary sea water.     

EFFECTS OF GUAIACOL AND HEXYLRESORCINOL IN THE PRESENCE OF BARIUM AND CALCIUM

Guaiacol was applied at two spots on the same cell of Nitella. At one spot it was dissolved in 0.01 M NaCl, at the other in 0.01 M CaCl₂ or BaCl₂. The effect was practically the same in all cases, i.e. a similar change of P.D. in a negative direction, involving a more or less complete loss of P.D. (depolarization). When hexylresorcinol was used in place of guaiacol the result was similar. That Ca⁺⁺ and Ba⁺⁺ do not inhibit the effect of these organic depolarizing substances may be due to a lack of penetration of Ca⁺⁺ and Ba⁺⁺. The organic substances penetrate more rapidly and their effect is chiefly on the inner protoplasmic surface which is the principal seat of the P.D.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : III. SOME EFFECTS OF GUAIACOL ON HALICYSTIS

Lowering the pH of sea water from 8.2 to 6.4 lowers the positive P.D. of Halicystis reversibly (this does not happen with Valonia). Exposure to sea water at pH 6.4 does not affect the apparent mobility of Na⁺ or of K⁺ (this agrees with Valonia). Guaiacol makes the P.D. of Halicystis less positive (in Valonia it has the opposite effect). Exposure to guaiacol does not reverse the effect of KCl in Halicystis which in this respect differs from Valonia. The P.D. can be changed from 66 mv. positive to 23 mv. negative by the combined action of KCl and guaiacol. Exposure to guaiacol affects Halicystis and Valonia similarly in respect to their behavior with dilute sea water. Normally the dilute sea water makes the P.D. more negative but after sufficient exposure to guaiacol dilute sea water either produces no change in P.D. or makes it more positive. In the latter case we may assume that the apparent mobility of Na⁺ has become greater than that of Cl^- as the result of the action of guaiacol. (Normally the apparent mobility of Cl^- is greater than that of Na⁺.) In Halicystis, as in Valonia and in Nitella, an organic substance can greatly change the apparent mobilities of certain inorganic ions (K⁺ or Na⁺).     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.     

A THEORY OF INJURY AND RECOVERY : II. EXPERIMENTS WITH MIXTURES.

1. The equations which serve to predict the injury of tissue in 0.52 M NaCl and in 0.278 M CaCl₂ and its subsequent recovery (when it is replaced in sea water) also enable us to predict the behavior of tissue in mixtures of these solutions, as well as its recovery in sea water after exposure to mixtures. 2. The reactions which are assumed in order to account for the behavior of the tissue proceed as if they were inhibited by a salt compound formed by the union of NaCl and CaCl₂ with some constituent of the protoplasm (certain of these reactions are accelerated by CaCl₂). 3. In this and preceding papers a quantitative theory is developed in order to explain: (a) the toxicity of NaCl and CaCl₂; (b) the antagonism between these substances; (c) the fact that recovery (in sea water) may be partial or complete, depending on the length of exposure to the toxic solution.     

NOTE ON THE NATURE OF THE CURRENT OF INJURY IN TISSUES

Leading off from two places on the same cell (of Nitella) with 0.001 M KCl we observe that a cut produces only a temporary negative current of injury. If we lead off with 0.001 M KCl from any cell to a neighboring cell we find that when sap comes out from the cut cell and reaches the neighboring intact cell a lasting negative "current of injury" is produced. This depends on the fact that the intact cell is in contact with sap at one point and with 0.001 M KCl at the other (this applies also to tissues composed of small cells). If we employ 0.1 M KCl in place of 0.001 M the current of injury with a single cell is positive (and is more lasting when a neighboring cell is present). Divergent results obtained with tissues and single cells may be due in part to these factors.     

THE INFLUENCE OF THE CHEMICAL NATURE OF SOLID PARTICLES ON THEIR CATAPHORETIC P.D. IN AQUEOUS SOLUTIONS

1. The effect of eight salts, NaCl, Na₂SO₄, Na₄Fe(CN)₆, CaCl₂, LaCl₃, ThCl₄, and basic and acid fuchsin on the cataphoretic P.D. between solid particles and aqueous solutions was measured near the point of neutrality of water (pH 5.8). It was found that without the addition of electrolyte the cataphoretic P.D. between particles and water is very minute near the point of neutrality (pH 5.8), often less than 10 millivolts, if care is taken that the solutions are free from impurities. Particles which in the absence of salts have a positive charge in water near the point of neutrality (pH 5.8) are termed positive colloids and particles which have a negative charge under these conditions are termed negative colloids. 2. If care is taken that the addition of the salt does not change the hydrogen ion concentration of the solution (which in these experiments was generally pH 5.8) it can be said in general, that as long as the concentration of salts is not too high, the anions of the salt have the tendency to make the particles more negative (or less positive) and that cations have the opposite effect; and that both effects increase with the increasing valency of the ions. As soon as a maximal P.D. is reached, which varies for each salt and for each type of particles, a further addition of salt depresses the P.D. again. Aside from this general tendency the effects of salts on the P.D. are typically different for positive and negative colloids. 3. Negative colloids (collodion, mastic, Acheson''s graphite, gold, and metal proteinates) are rendered more negative by low concentrations of salts with monovalent cation (e.g. Na) the higher the valency of the anion, though the difference in the maximal P.D. is slight for the monovalent Cl and the tetravalent Fe(CN)₆ ions. Low concentrations of CaCl₂ also make negative colloids more negative but the maximal P.D. is less than for NaCl; even LaCl₃ increases the P.D. of negative particles slightly in low concentrations. ThCl₄ and basic fuchsin, however, seem to make the negative particles positive even in very low concentrations. 4. Positive colloids (ferric hydroxide, calcium oxalate, casein chloride—the latter at pH 4.0) are practically not affected by NaCl, are rendered slightly negative by high concentrations of Na₂SO₄, and are rendered more negative by Na₄Fe(CN)₆ and acid dyes. Low concentrations of CaCl₂ and LaCl₃ increase the positive charge of the particles until a maximum is reached after which the addition of more salt depresses the P.D. again. 5. It is shown that alkalies (NaOH) act on the cataphoretic P.D. of both negative and positive particles as Na₄Fe(CN)₆ does at the point of neutrality. 6. Low concentrations of HCl raise the cataphoretic P.D. of particles of collodion, mastic, graphite, and gold until a maximum is reached, after which the P.D. is depressed by a further increase in the concentration of the acid. No reversal in the sign of charge of the particle occurs in the case of collodion, while if a reversal occurs in the case of mastic, gold, and graphite, the P.D. is never more than a few millivolts. When HCl changes the chemical nature of the colloid, e.g. when HCl is added to particles of amphoteric electrolytes like sodium gelatinate, a marked reversal will occur, on account of the transformation of the metal proteinate into a protein-acid salt. 7. A real reversal in the sign of charge of positive particles occurs, however, at neutrality if Na₄Fe(CN)₆ or an acid dye is added; and in the case of negative colloids when low concentrations of basic dyes or minute traces of ThCl₄ are added. 8. Flocculation of the suspensions by salts occurs when the cataphoretic P.D. reaches a critical value which is about 14 millivolts for particles of graphite, gold, or mastic or denatured egg albumin; while for collodion particles it was about 16 millivolts. A critical P.D. of about 15 millivolts was also observed by Northrop and De Kruif for the flocculation of certain bacteria.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : II. THE EFFECTS OF POTASSIUM ON TWO SPECIES WITH DIFFERENT SAPS

The potential difference across the protoplasm of impaled cells of two American species of Halicystis is compared. The mean value for H. Osterhoutii is 68.4 mv.; that for H. ovalis is 79.7 mv., the sea water being positive to the sap in both. The higher potential of H. ovalis is apparently due to the higher concentration of KCl (0.3 M) in its vacuolar sap. When the KCl content of H. Osterhoutii sap (normally 0.01 M or less) is experimentally raised to 0.3 M, the potential rises to values about equal to those in H. ovalis. The external application of solutions high in potassium temporarily lowers the potential of both, probably by the high mobility of K⁺ ions. But a large potential is soon regained, representing the characteristic potential of the protoplasm. This is about 20 mv. lower than in sea water. The accumulation of KCl in the sap of H. ovalis is apparently not due to the higher mobility of K⁺ ion in its protoplasm, since the electrical effects of potassium are practically identical in H. Osterhoutii, where KCl is not accumulated.     

THE NUCLEOPROTAMINE OF TROUT SPERM

The nucleoprotamine of trout sperm can be extracted completely with 1 M sodium chloride. On reducing the salt concentration to 0.14 M, physiological saline, the nucleoprotamine precipitates in long, fibrous strands. When the nucleoprotamine, dissolved in M NaCl, is dialyzed all the protamine diffuses through the membrane leaving behind highly polymerized, protein-free desoxyribose nucleic acid. The nucleoprotamine constitutes 91 per cent of the lipid-free mass of the sperm nucleus. While nucleoprotamine is being extracted by M NaCl a stage is reached at which the sperm chromosomes are clearly visible.