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.     

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.     

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.     

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.     

EFFECTS OF HEXYLRESORCINOL ON NITELLA

In some ways the effects of hexylresorcinol on Nitella resemble those of guaiacol but in others they differ. Both substances depress the P.D. reversibly and both decrease the potassium effect. Hexylresorcinol decreases the apparent mobility of Na⁺ and of K⁺. Guaiacol increases that of Na⁺ but not of K⁺. The action of hexylresorcinol is more striking than that of guaiacol since 0.0003 M of the former is as effective as 0.03 M of the latter in depressing the P.D. It is evident that organic substances can change the behavior of inorganic ions in a variety of ways.     

THE PHYSIOLOGICAL EFFECTS OF l-TYROSINE AND l-PHENYL-ALANINE ON THE RATE AND QUALITY OF PULSATION IN THE SEVENTY-TWO HOUR EXTIRPATED EMBRYONIC CHICK HEART

1. 72 hour isolated chick hearts show an increase in pulsation rate when placed in M/1000, M/10,000, and M/50,000 l-tyrosine solutions. The optimal effect is seen in M/10,000 and M/50,000 l-tyrosine. 2. All hearts show disturbance of rhythm either in the form of irregular rhythm or heart block. 3. 62 hour isolated chick hearts are not susceptible to l-tyrosine while 96 hour hearts are markedly sensitive. 4. 72 hour isolated chick hearts placed in 1 part in 10,000 and 1 part in 50,000 l-epinephrine show approximately the same effects as were seen with l-tyrosine. 5. 72 hour isolated chick hearts placed in M/1000 and M/10,000 l-phenylalanine show an initial depression followed by an l-tyrosine effect.     

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^-.     

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.     

EFFECTS OF HYDROXYL ON NEGATIVE AND POSITIVE CELLS OF NITELLA

Remarkable changes are brought about by KOH in transforming negative cells of Nitella (showing dilute solution negative with KOH) to positive cells (showing dilute solution positive with KOH). NaOH is less effective as a transforming agent. This might be explained on the ground that the protoplasm contains an acid (possibly a fatty acid) which makes the cell negative and which is dissolved out more rapidly by KOH than by NaOH, as happens with the fatty acids in ordinary soaps. Part of a negative cell can be changed to positive by exposure to KOH while the untreated portion remains negative. After exposure to KOH the potential the protoplasm has when in contact with NaCl may increase. At the same time there may be an increase in the potassium effect; i.e., in the change of P.D. in a positive direction observed when 0.01 M KCl is replaced by 0.01 M NaCl. In some cases the order of ionic mobilities is u _K > v _OH > u _Na. This shows that the protoplasmic surface cannot be a pore system: for in such a system all cations must have greater mobilities than all anions or vice versa.     

THE ACCUMULATION OF ELECTROLYTES : IV. INTERNAL VERSUS EXTERNAL CONCENTRATIONS OF POTASSIUM

Lowering the potassium in the sea water from 0.011 M to 0.006 M caused an exit of potassium from cells of Valonia macrophysa. Sodium continued to penetrate and the ratio K ÷ Na fell off. The cells ceased to grow but there was no evidence of injury. Increasing the external potassium brought about an increase of the internal concentration of potassium, of halide, of total cations, and of the ratio K ÷ Na inside. These phenomena are to be expected on theoretical grounds.     

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.     

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.     

DELAYED POTASSIUM EFFECT IN NITELLA

In normal cells of Nitella replacement of NaCl by KCl makes the P.D. much less positive: this is called the potassium effect. Cells which have lost the potassium effect usually show little or no change of P.D. when NaCl is replaced by KCl but an occasional cell responds after a delay. It seems possible that the delay may be largely due to the time required for potassium to combine with an organic substance, thus forming a compound which sensitizes the protoplasmic surface to the action of potassium.     

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.     

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.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : III. OXYGEN CONSUMPTION AND CELL DIVISION OF FERTILIZED SEA URCHIN EGGS IN THE PRESENCE OF RESPIRATORY INHIBITORS

1. The effects of a number of respiratory inhibiting agents on the cell division of fertilized eggs of Arbacia punctulata have been determined. For eggs initially exposed to the reagents at 30 minutes after fertilization at 20°C., the levels of oxygen consumption prevailing in the minimum concentrations of reagents which produced complete cleavage block were (as percentages of the control): In 0.4 per cent O₂-99.6 per cent N₂, 32; in 0.7 per cent O₂-99.3 per cent CO, 32; in 1.6 x 10^–4 M potassium cyanide, 34; in 1 x 10^–3 M phenylurethane, 70; in 4 x 10^–3 M 5-isoamyl-5-ethyl barbituric acid, 20; in 3 x 10^–4 M iodoacetic acid, 53. 2. The carbon monoxide inhibition of oxygen consumption and cell division was reversed by light. The percentage inhibition of oxygen consumption by carbon monoxide in the dark is described by the usual mass action equation with K, the inhibition constant, equal to approximately 60, as compared to values of 5 to 10 for yeast and muscle. In 20 per cent O₂-80 per cent CO in the dark there was a slight stimulation of oxygen consumption, averaging 20 per cent. 3. Spectroscopic examination of fertilized and unfertilized Arbacia eggs reduced by hydrosulfite revealed no cytochrome bands. The thickness and density of the egg suspension was such as to indicate that, if cytochrome is present at all, the amount in Arbacia eggs is extremely small as compared to that in other tissues having a comparable rate of oxygen consumption. 4. Three reagents poisoning copper catalyses, potassium dithio-oxalate (10^–2 M), diphenylthiocarbazone (10^–4 M), and isonitrosoacetophenone (2 x 10^–3 M) produced no inhibition of division of fertilized Arbacia eggs. 5. These results indicate that the respiratory processes required to support division in the Arbacia egg may perhaps differ in certain essential steps from the principal respiratory processes in yeast and muscle.     

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 EFFECTS OF CAFFEINE ON OXYGEN CONSUMPTION AND CELL DIVISION IN THE FERTILIZED EGG OF THE SEA URCHIN,ARBACIA PUNCTULATA

1. By means of the Warburg-Barcroft microrespirometer apparatus and the Warburg direct method, the relative effect of caffeine upon the O₂ consumption of the fertilized egg of Arbacia punctulata was shown for the following concentrations in sea water: 0.002 per cent (M/10,000), 0.004 per cent (M/5,000), 0.02 per cent (M/1,000), 0.1 per cent (M/200), 0.2 per cent (M/100), 0.5 per cent (M/40), and 2 per cent (M/10). 2. In comparison with the normal eggs (uninhibited, non-caffeine-treated controls), caffeine in concentrations including and greater than 0.1 per cent (M/200) depressed the average uptake from approximately 25 to 61 per cent over the 3 hour period. In a number of instances, as typified by Experiment 10, the effective inhibitory concentration ranged from 0.02 per cent (M/1,000) upward and the degree of depression of the O₂ consumption ranged from 10.6 per cent to 60.6 per cent. 3. All caffeine concentrations including and above 0.02 per cent (M/1,000) in the series used, resulted in decreasing the normal rate of cleavage division in the fertilized Arbacia eggs. 4. The higher concentrations (0.5 and 2 per cent) produced a complete blockage of the cleavage process. 5. Complete cleavage inhibition was noted only when the O₂ uptake had been depressed to 50 per cent or more of the normal controls. 6. O₂ consumption-time relationship data indicate an average depression, in O₂ consumption over a 3 hour period, ranging from 25 per cent with a caffeine concentration of 0.1 per cent to a 61 per cent inhibition with a concentration of 2 per cent. 7. Concentrations of less than 0.1 per cent (certainly of less than 0.02 per cent) give variable results and indicate no significant effect. 8. It is inferred from the respiration data presented that it is probable that the inhibition of the O₂ consumption in fertilized Arbacia eggs is due to the influence of caffeine upon the main (activity or primary) pathway. It will be observed that there are certain similarities of the caffeine data to the degree of inhibition accomplished by sodium cyanide. Moreover, it has been demonstrated that the cyanide probably acts on the cytochrome oxidase step in the cytochrome oxidase-cytochrome chain of reactions constituting the O₂ uptake phase of respiratory metabolism. It is not improbable, therefore, that caffeine also may act upon the cytochrome oxidase enzyme. 9. From the viewpoint of environmental conditions influencing reproductive phenomena, it is of interest that caffeine can affect the normal metabolism of the zygote.     

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 : 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.