THE COLLOIDAL BEHAVIOR OF PROTEINS

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

THE ORIGIN OF THE ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND OF LIVING TISSUES

1. When a solution of a salt of gelatin or crystalline egg albumin is separated by a collodion membrane from a watery solution (free from protein) a potential difference is set up across the membrane in which the protein is positively charged in the case of protein-acid salts and in which the protein is negatively charged in the case of metal proteinates. The turning point is the isoelectric point of the protein. 2. Measurements of the pH of the (inside) protein solution and of the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive in the case of protein-acid salts and negative in the case of metal proteinates. This is to be expected when the P.D. is caused by the establishment of a Donnan equilibrium, since in that case the pH should be lower outside than inside in the case of a protein-acid salt and should be higher outside than inside in the case of a metal proteinate. 3. At the isoelectric point where the electrical charge is zero the value of pH inside minus pH outside becomes also zero. 4. It is shown that a P.D. is established between suspended particles of powdered gelatin and the surrounding watery solution and that the sign of charge of the particles is positive when they contain gelatin-acid salts, while it is negative when the powdered particles contain metal gelatinate. At the isoelectric point the charge is zero. 5. Measurements of the pH inside the powdered particles and of the pH in the outside watery solution show that when equilibrium is established the value pH inside minus pH outside is positive when the powdered particles contain a gelatin-acid salt, while the value pH inside minus pH outside is negative when the powdered particles contain Na gelatinate. At the isoelectric point the value pH inside minus pH outside is zero. 6. The addition of neutral salts depresses the electrical charge of the powdered particles of protein-acid salts. It is shown that the addition of salts to a suspension of powdered particles of gelatin chloride also diminishes the value of pH inside minus pH outside. 7. The agreement between the values 58 (pH inside minus pH outside) and the P. D. observed by the Compton electrometer is not only qualitative but quantitative. This proves that the difference in the concentration of acid (or alkali, as the case may be) in the two phases is the only cause for the observed P.D. 8. The Donnan theory demands that the P.D. of a gelatin chloride solution should be 1½ times as great as the P.D. of a gelatin sulfate solution of the same pH and the same concentration (1 per cent) of originally isoelectric gelatin. This is found to be correct and it is also shown that the values of pH inside minus pH outside for the two solutions possess the ratio of 3:2. 9. All these measurements prove that the electrical charges of suspended particles of protein are determined exclusively by the Donnan equilibrium.     

THE ELIMINATION OF DISCREPANCIES BETWEEN OBSERVED AND CALCULATED P.D. OF PROTEIN SOLUTIONS NEAR THE ISOELECTRIC POINT WITH THE AID OF BUFFER SOLUTIONS

1. It had been noticed in the previous experiments on the influence of the hydrogen ion concentration on the P.D. between protein solutions inside a collodion bag and aqueous solutions free from protein that the agreement between the observed values and the values calculated on the basis of Donnan''s theory was not satisfactory near the isoelectric point of the protein solution. It was suspected that this was due to the uncertainty in the measurements of the pH of the outside aqueous solution near the isoelectric point. This turned out to be correct, since it is shown in this paper that the discrepancy disappears when both the inside and outside solutions contain a buffer salt. 2. This removes the last discrepancy between the observed P.D. and the P. D. calculated on the basis of Donnan''s theory of P.D. between membrane equilibria, so that we can state that the P.D. between protein solutions inside collodion bags and outside aqueous solutions free from protein can be calculated from differences in the hydrogen ion concentration on the opposite sides of the membrane, in agreement with Donnan''s formula.     

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

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

THE INFLUENCE OF 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.     

CATAPHORETIC CHARGES OF COLLODION PARTICLES AND ANOMALOUS OSMOSIS THROUGH COLLODION MEMBRANES FREE FROM PROTEIN

1. It had been shown in previous papers that when a salt solution is separated from pure water by a collodion membrane, water diffuses through the membrane as if it were positively charged and as if it were attracted by the anion of the salt in solution and repelled by the cation with a force increasing with the valency. In this paper, measurements of the P.D. across the membrane (E) are given, showing that when an electrical effect is added to the purely osmotic effect of the salt solution in the transport of water from the side of pure water to the solution, the latter possesses a considerable negative charge which increases with increasing valency of the anion of the salt and diminishes with increasing valency of the cation. It is also shown that a similar valency effect exists in the diffusion potentials between salt solutions and pure water without the interposition of a membrane. 2. This makes it probable that the driving force for the electrical transport of water from the side of pure water into solution is primarily a diffusion potential. 3. It is shown that the hydrogen ion concentration of the solution affects the transport curves and the diffusion potentials in a similar way. 4. It is shown, however, that the diffusion potential without interposition of the membrane differs in a definite sense from the P.D. across the membrane and that therefore the P.D. across the membrane (E) is a modified diffusion potential. 5. Measurements of the P.D. between collodion particles and aqueous solutions (ε) were made by the method of cataphoresis, which prove that water in contact with collodion particles free from protein practically always assumes a positive charge (except in the presence of salts with trivalent and probably tetravalent cations of a sufficiently high concentration). 6. It is shown that an electrical transport of water from the side of water into the solution is always superposed upon the osmotic transport when the sign of charge of the solution in the potential across the membrane (E) is opposite to that of the water in the P.D. between collodion particle and water (ε); supporting the theoretical deductions made by Bartell. 7. It is shown that the product of the P.D. across the membrane (E) into the cataphoretic P.D. between collodion particles and aqueous solution (ε) accounts in general semiquantitatively for that part of the transport of water into the solution which is due to the electrical forces responsible for anomalous osmosis.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS

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

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

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

THE INFLUENCE OF ELECTROLYTES ON THE CATAPHORETIC CHARGE OF COLLOIDAL PARTICLES AND THE STABILITY OF THEIR SUSPENSIONS : I. EXPERIMENTS WITH COLLODION PARTICLES.

1. When collodion particles suspended in water move in an electric field they are, as a rule, negatively charged. The maximal cataphoretic P.D. between collodion particles and water is about 70 millivolts. This is only slightly more than the cataphoretic P.D. found by McTaggart to exist between gas bubbles and water (55 millivolts). Since in the latter case the P.D. is entirely due to forces inherent in the water itself, resulting possibly in an excess of OH ions in the layer of water in contact and moving with the gas bubble, it is assumed that the negative charge of the collodion particles is also chiefly due to the same cause; the collodion particles being apparently only responsible for the slight difference in maximal P.D. of water-gas and water-collodion surfaces. 2. The cataphoretic charge of collodion particles seems to be a minimum in pure water, increasing as a rule with the addition of electrolytes, especially if the cation of the electrolyte is monovalent, until a maximal P.D. is reached. A further increase in the concentration of the electrolyte depresses the P.D. again. There is little difference in the action of HCl, NaOH, and NaCl or LiCl or KCl. 3. The increase in P.D. between collodion particles and water upon the addition of electrolyte is the more rapid the higher the valency of the anion. This suggests that this increase of negative charge of the collodion particle is due to the anions of the electrolyte gathering in excess in the layer of water nearest to the collodion particles, while the adjoining aqueous layer has an excess of cations. 4. In the case of chlorides and at a pH of about 5.0 the maximal P.D. between collodion particles and water is about 70 millivolts, when the cation of the electrolyte present is monovalent (H, Li, Na, K); when the cation of the electrolyte is bivalent (Mg, Ca), the maximal P.D. is about 35 to 40 millivolts; and when the cation is trivalent (La) the maximal P.D. is lower, probably little more than 20 millivolts. 5. A reversal in the sign of charge of the collodion particles could be brought about by LaCl₃ but not by acid. 6. These results on the influence of electrolytes on the cataphoretic P.D. between collodion particles and water are also of significance for the theory of electrical endosmose and anomalous osmosis through collodion membranes; since the cataphoretic P.D. is probably identical with the P.D. between water and collodion inside the pores of a collodion membrane through which the water diffuses. 7. The cataphoretic P.D. between collodion particles and water determines the stability of suspensions of collodion particles in water, since rapid precipitation occurs when this P.D. falls below a critical value of about 16 millivolts, regardless of the nature of the electrolyte by which the P.D. is depressed. No peptization effect of plurivalent anions was noticed.     

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

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

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.     

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.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VIII. THE EFFECTS OF LIGHT

The effects of light upon the potential difference across the protoplasm of impaled Halicystis cells are described. These effects are very slight upon the normal P.D., increasing it 3 or 4 per cent, or at most 10 per cent, with a characteristic cusped time course, and a corresponding decrease on darkening. Light effects become much greater when the P.D. has been decreased by low O₂ content of the sea water; light restores the P.D. in much the same time course as aeration, and doubtless acts by the photosynthetic production of O₂. There are in both cases anomalous cusps which decrease the P.D. before it rises. Short light exposures may give only this anomaly. Over part of the potential range the light effects are dependent upon intensity. Increased CO₂ content of the sea water likewise depresses the P.D. in the dark, and light overcomes this depression if it is not carried too far. Recovery is probably due to photosynthetic consumption of CO₂, unless there is too much present. Again there are anomalous cusps during the first moments of illumination, and these may be the only effect if the P.D. is too low. The presence of ammonium salts in the sea water markedly sensitizes the cells to light. Subthreshold NH₄ concentrations in the dark become effective in the light, and the P.D. reverses to a negative sign on illumination, recovering again in the dark. This is due to increase of pH outside the cell as CO₂ is photosynthetically reduced, with increase of undissociated NH₃ which penetrates the cell. Anomalous cusps which first carry the P.D. in the opposite direction to the later drift are very marked in the presence of ammonia, and may represent an increased acidity which precedes the alkaline drift of photosynthesis. This acid gush seems to be primarily within the protoplasm, persisting when the sea water is buffered. Glass electrode measurements also indicate anomalies in the pH drift. There are contrary cusps on darkening which suggest temporarily increased alkalinity. Even more complex time courses are given by combining low O₂ and NH₄ exposures with light; these may have three or more cusps, with reversal, recovery, and new reversal. The ultimate cause of the light effects is to be found in an alteration of the surface properties by the treatments, which is overcome (low O₂, high CO₂), or aided (NH₄) by light. This alteration causes the surface to lose much of its ionic discrimination, and increases its electrical resistance. Tests with various anion substitutions indicate this, with recovery of normal response in the light. A theory of the P.D. in Halicystis is proposed, based on low mobility of the organic anions of the protoplasm, with differences in the two surfaces with respect to these, and the more mobile Na and K. ions.     

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 MECHANISM BY WHICH TRIVALENT AND TETRAVALENT IONS PRODUCE AN ELECTRICAL CHARGE ON ISOELECTRIC PROTEIN

1. Experiments on anomalous osmosis suggested that salts with trivalent cations, e.g. LaCl₃, caused isoelectric gelatin to be positively charged, and salts with tetravalent anions, e.g. Na₄Fe(CN)₆, caused isoelectric gelatin to be negatively charged. In this paper direct measurements of the P.D. between gels of isoelectric gelatin and an aqueous solution as well as between solutions of isoelectric gelatin in a collodion bag and an aqueous solution are published which show that this suggestion was correct. 2. Experiments on anomalous osmosis suggested that salts like MgCl₂, CaCl₂, NaCl, LiCl, or Na₂SO₄ produce no charge on isoelectric gelatin and it is shown in this paper that direct measurements of the P.D. support this suggestion. 3. The question arose as to the nature of the mechanism by which trivalent and tetravalent ions cause the charge of isoelectric proteins. It is shown that salts with such ions act on isoelectric gelatin in a way similar to that in which acids or alkalies act, inasmuch as in low concentrations the positive charge of isoelectric gelatin increases with the concentration of the LaCl₃ solution until a maximum is reached at a concentration of LaCl₃ of about M/8,000; from then on a further increase in the concentration of LaCl₃ diminishes the charge again. It is shown that the same is true for the action of Na₄Fe(CN)₆. From this it is inferred that the charge of the isoelectric gelatin under the influence of LaCl₃ and Na₄Fe(CN)₆ at the isoelectric point is due to an ionization of the isoelectric protein by the trivalent or tetravalent ions. 4. This ionization might be due to a change of the pH of the solution, but experiments are reported which show that in addition to this influence on pH, LaCl₃ causes an ionization of the protein in some other way, possibly by the formation of a complex cation, gelatin-La. Na₄Fe(CN)₆ might probably cause the formation of a complex anion of the type gelatin-Fe(CN)₆. Isoelectric gelatin seems not to form such compounds with Ca, Na, Cl, or SO₄. 5. Solutions of LaCl₃ and Na₄Fe(CN)₆ influence the osmotic pressure of solutions of isoelectric gelatin in a similar way as they influence the P.D., inasmuch as in lower concentrations they raise the osmotic pressure of the gelatin solution until a maximum is reached at a concentration of about M/2,048 LaCl₃ and M/4,096 Na₄Fe(CN)₆. A further increase of the concentration of the salt depresses the osmotic pressure again. NaCl, LiCl, MgCl₂, CaCl₂, and Na₂SO₄ do not act in this way. 6. Solutions of LaCl₃ have only a depressing effect on the P.D. and osmotic pressure of gelatin chloride solutions of pH 3.0 and this depressing effect is quantitatively identical with that of solutions of CaCl₂ and NaCl of the same concentration of Cl.     

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^-.     

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.     

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.     

BIOELECTRIC POTENTIALS IN HALICYSTIS : VII. THE EFFECTS OF LOW OXYGEN TENSION

The potential difference across the protoplasm of impaled cells of Halicystis is not affected by increase of oxygen tension in equilibrium with the sea water, nor with decrease down to about 1/10 its tension in the air (2 per cent O₂ in N₂). When bubbling of 2 per cent O₂ is stopped, the P.D. drifts downward, to be restored on stirring the sea water, or rebubbling the gas. Bubbling 0.2 per cent O₂ causes the P.D. to drop to 20 mv. or less; 1.1 per cent O₂ to about 50 mv. Restoration of 2 per cent or higher O₂ causes recovery to 70 or 80 mv. often with a preliminary cusp which decreases the P.D. before it rises. Perfusion of aerated sea water through the vacuole is just as effective in restoring the P.D. as external aeration, indicating that the direction of the oxygen gradient is not significant. Low O₂ tension also inhibits the reversed, negative P.D. produced by adding NH₄Cl to sea water, 0.2 per cent O₂ bringing this P.D. back to the same low positive values found without ammonia. Restoration of 2 per cent O₂ or air, restores this latent negativity. At slightly below the threshold for ammonia reversal, low O₂ may induce a temporary negativity when first bubbled, and a negative cusp may occur on aeration before positive P.D. is regained. This may be due to a decreased consumption of ammonia, or to intermediate pH changes. The locus of the P.D. alteration was tested by applying increased KCl concentrations to the cell exterior; the large cusps produced in aerated solutions become greatly decreased when the P.D. has fallen in 0.2 per cent O₂. This indicates that the originally high relative mobility or concentration of K⁺ ion has approached that of Na⁺ in the external protoplasmic surface under reduced O₂ tension. Results obtained with sulfate sea water indicate that Na⁺ mobility approaches that of SO₄ ^— in 0.2 per cent O₂. P.D. measurements alone cannot tell whether this is due to an increase of the slower ion or a decrease of the faster ion. A decrease of all ionic permeability is indicated, however, by a greatly increased effective resistance to direct current during low O₂. Low resistance is regained on aeration. The resistance increase resembles that produced by weak acids, cresol, etc. Acids or other substances produced in anaerobiosis may be responsible for the alteration. Or a deficiency of some surface constituent may develop. In addition to the surface changes there may be alterations in gradients of inorganic or organic ions within the protoplasm, but there is at present no evidence on this point. The surface changes are probably sufficient to account for the phenomena.