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.     

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.     

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.     

INFLUENCE OF A SLIGHT MODIFICATION OF THE COLLODION MEMBRANE ON THE SIGN OF THE ELECTRIFICATION OF WATER

1. It is shown that collodion membranes which have received one treatment with a 1 per cent gelatin solution show for a long time (if not permanently) afterwards a different osmotic behavior from collodion membranes not treated with gelatin. This difference shows itself only towards solutions of those electrolytes which have a tendency to induce a negative electrification of the water particles diffusing through the membrane, namely solutions of acids, acid salts, and of salts with trivalent and tetravalent cations; while the osmotic behavior of the two types of membranes towards solutions of salts and alkalies, which induce a positive electrification of the water particles diffusing through the membrane, is the same. 2. When we separate solutions of salts with trivalent cation, e.g. LaCl₃ or AlCl₃, from pure water by a collodion membrane treated with gelatin, water diffuses rapidly into the solution; while no water diffuses into the solution when the collodion membrane has received no gelatin treatment. 3. When we separate solutions of acid from pure water by a membrane previously treated with gelatin, negative osmosis occurs; i.e., practically no water can diffuse into the solution, while the molecules of solution and some water diffuse out. When we separate solutions of acid from pure water by collodion membranes not treated with gelatin, positive osmosis will occur; i.e., water will diffuse rapidly into the solution and the more rapidly the higher the valency of the anion. 4. These differences occur only in that range of concentrations of electrolytes inside of which the forces determining the rate of diffusion of water through the membrane are predominantly electrical; i.e., in concentrations from 0 to about M/16. For higher concentrations of the same electrolytes, where the forces determining the rate of diffusion are molecular, the osmotic behavior of the two types of membranes is essentially the same. 5. The differences in the osmotic behavior of the two types of membranes are not due to differences in the permeability of the membranes for solutes since it is shown that acids diffuse with the same rate through both kinds of membranes. 6. It is shown that the differences in the osmotic behavior of the two types of collodion membranes towards solutions of acids and of salts with trivalent cation are due to the fact that in the presence of these electrolytes water diffuses in the form of negatively charged particles through the membranes previously treated with gelatin, and in the form of positively charged particles through collodion membranes not treated with gelatin. 7. A treatment of the collodion membranes with casein, egg albumin, blood albumin, or edestin affects the behavior of the membrane towards salts with trivalent or tetravalent cations and towards acids in the same way as does a treatment with gelatin; while a treatment of the membranes with peptone prepared from egg albumin, with alanine, or with starch has no such effect.     

STABILITY OF SUSPENSIONS OF SOLID PARTICLES OF PROTEINS AND PROTECTIVE ACTION OF COLLOIDS

1. It is shown that the concentrations of different salts required to precipitate suspensions of gelatin-coated collodion particles in water are practically identical with the concentrations of the same salts required for the "salting out" of gelatin from aqueous solutions. Neither effect shows any relation to the electrical double layers surrounding the particles. 2. It is shown that at the isoelectric point of gelatin, suspensions of gelatin-coated collodion particles are not stable and it had been shown previously that gelatin is least soluble at the isoelectric point. The addition of salt increases both the solubility of gelatin in water as well as the stability of suspensions of gelatin-coated collodion particles in water, and both effects increase with the valency of one of the ions of the salt. 3. This latter effect is not due to any charges conferred on the gelatin particles by the salts, since the cataphoretic experiments show that salts like NaCl, Na₂SO₄, or CaCl₂, which at the isoelectric point of gelatin increase the solubility of gelatin as well as the stability of suspensions of gelatin-coated collodion particles, leave the particles practically uncharged in the concentrations in which the salts are efficient. 4. It follows from all these facts that the stability of suspensions of gelatin-coated particles in water depends on the solubility of gelatin in water; e.g., on the chemical affinity of certain groups of the gelatin molecule for water. 5. Though crystalline egg albumin is highly soluble in water, the stability of collodion particles coated with crystalline egg albumin does not depend upon the affinity of the albumin molecule for water, but depends practically alone on the electrical double layer surrounding each particle. As soon as the P.D. of this double layer falls below 13 millivolts, the suspension is no longer stable. 6. The critical potential for the stability of suspensions of collodion particles coated with genuine egg albumin is the same as that for particles of boiled (denatured) white of egg. Since through the process of heating, egg albumin loses its solubility in water, it is inferred that egg albumin undergoes the same change when it forms a film around a solid particle like collodion. 7. The influence of electrolytes on the stability of suspensions of collodion particles coated with casein or edestin was similar to that of collodion particles coated with egg albumin. The experiments are, however, complicated by the fact that near the isoelectric point CaCl₂ and even NaCl cause a suspension again at concentrations of about M/2 or 1 M, while still higher concentrations may cause a precipitation again. These latter effects have no connection with double layers, but belong probably in the category of solubility phenomena. 8. These experiments permit us to define more definitely the conditions for a general protective action of colloids. Protective colloids must be capable of forming a durable film on the surface of the suspended particles and the molecules constituting the film must have a higher attraction for the molecules of the solvent than for each other; in other words, they must possess true solubility. Only in this case can they prevent the precipitating action of low concentrations of electrolytes on particles which are kept in suspension solely by the high potentials of an electrical double layer. Thus gelatin films, in which the attraction of the molecules for water is preserved, have a general protective action, while crystalline egg albumin, casein, and edestin, which seem to lose their attraction for water when forming a film, have a protective action only under limited conditions stated in the paper.     

ON THE LOCATION OF THE FORCES WHICH DETERMINE THE ELECTRICAL DOUBLE LAYER BETWEEN COLLODION PARTICLES AND WATER

1. The cataphoretic P.D. of suspended particles is assumed to be due to an excess in the concentration of one kind of a pair of oppositely charged ions in the film of water enveloping the particles and this excess is generally ascribed to a preferential adsorption of this kind of ions by the particle. The term adsorption fails, however, to distinguish between the two kinds of forces which can bring about such an unequal distribution of ions between the enveloping film and the opposite film of the electrical double layer, namely, forces inherent in the water itself and forces inherent in the particle (e.g. chemical attraction between particle and adsorbed ions). 2. It had been shown in a preceding paper that collodion particles suspended in an aqueous solution of an ordinary electrolyte like NaCl, Na₂SO₄, Na₄Fe(CN)₆, CaCl₂, HCl, H₂SO₄, or NaOH are always negatively charged, and that the addition of these electrolytes increases the negative charge as long as their concentration is below M/1,000 until a certain maximal P.D. is reached. Hence no matter whether acid, alkali, or a neutral salt is added, the concentration of anions must always be greater in the film enveloping the collodion particles than in the opposite film of the electrical double layer, and the reverse is true for the concentration of cations. This might suggest that the collodion particles, on account of their chemical constitution, attract anions with a greater force than cations, but such an assumption is rendered difficult in view of the following facts. 3. Experiments with dyes show that at pH 5.8 collodion particles are stained by basic dyes (i.e. dye cations) but not by acid dyes (i.e. dye anions), and that solutions of basic dyes are at pH 5.8 more readily decolorized by particles of collodion than acid dyes. It is also shown in this paper that crystalline egg albumin, gelatin, and Witte''s peptone form durable films on collodion only when the protein exists in the form of a cation or when it is isoelectric, but not when it exists in the form of an anion (i.e. on the alkaline side of its isoelectric point). Hence if any ions of dyes or proteins are permanently bound at the surface of collodion particles through forces inherent in the collodion they are cations but not anions. The fact that isoelectric proteins form durable films on collodion particles suggests, that the forces responsible for this combination are not ionic. 4. It is shown that salts of dyes or proteins, the cations of which are capable of forming durable films on the surface of the collodion, influence the cataphoretic P.D. of the collodion particles in a way entirely different from that of any other salts inasmuch as surprisingly low concentrations of salts, the cation of which is a dye or a protein, render the negatively charged collodion particles positive. Crystalline egg albumin and gelatin have such an effect even in concentrations of 1/130,000 or 1/65,000 of 1 per cent, i.e. in a probable molar concentration of about 10^–9. 5. Salts in which the dye or protein is an anion have no such effect but act like salts of the type of NaCl or Na₂SO₄ on the cataphoretic P.D. of collodion particles. 6. Amino-acids do not form durable films on the surface of collodion particles at any pH and the salts of amino-acids influence their cataphoretic P.D. in the same way as NaCl but not in the same way as proteins or dyes, regardless of whether the amino-acid ion is a cation or an anion. 7. Ordinary salts like LaCl₃ also fail to form a durable film on the surface of collodion particles. 8. Until evidence to the contrary is furnished, these facts seem to suggest that the increase of the negative charge of the collodion particles caused by the addition of low concentrations of ordinary electrolytes is chiefly if not entirely due to forces inherent in the aqueous solution but to a less extent, if at all, due to an attraction of the anions of the electrolyte by forces inherent in the collodion particles.     

MEMBRANE POTENTIALS AND CATAPHORETIC POTENTIALS OF PROTEINS

1. It has been shown in preceding publications that the membrane potentials of protein solutions or gels are determined by differences in the concentration of a common ion (e.g. hydrogen ion) inside a protein solution or protein gel and an outside aqueous solution free from protein, and that the membrane potentials can be calculated with a good degree of accuracy from Donnan''s equation for membrane equilibria. 2. On the basis of the theory of electrical double layers developed by Helmholtz, we are forced to assume that the cataphoretic potentials of protein particles are determined by a difference in the concentration of the two oppositely charged ions of the same electrolyte in the two strata of an electrical double layer surrounding the protein particle but situated entirely in the aqueous solution. 3. The membrane potentials of proteins agree with the cataphoretic potentials in that the sign of charge of the protein is negative on the alkaline side and positive on the acid side of the isoelectric point of the protein in both membrane potentials and cataphoretic potentials. The two types of potential of proteins disagree, especially in regard to the action of salts with trivalent and tetravalent ions on the sign of charge of the protein. While low concentrations of these salts bring about a reversal of the sign of the cataphoretic potentials of protein particles (at least in the neighborhood of the isoelectric point), the same salts can bring the membrane potentials of proteins only to zero, but call bring about no or practically no reversal of the sign of charge of the protein. Where salts seem to bring about a reversal in the membrane potential of protein solutions, the reversal is probably in reality always due to a change in the pH. 4. We may state, as a result of our experiments, that the cataphoretic migration and the cataphoretic P.D. of protein particles or of suspended particles coated with a protein are the result of two groups of forces; namely, first, forces inherent in the protein particles (these forces being linked with the membrane equilibrium between protein particles and the outside aqueous solution); and second, forces inherent entirely in the aqueous solution surrounding the protein particles. The forces inherent in the protein particles and linked with the membrane equilibrium prevail to such an extent over the forces inherent in the water, that the sense of the cataphoretic migration of protein particles is determined by the forces resulting from the membrane equilibrium.     

Thermal Properties of Freeze-Concentrated Sugar-Phosphate Solutions

In order to understand the effect of phosphate salts on the freeze-concentrated glass-like transition temperature (T _g′) of aqueous sugar solutions, two types of sugar (glucose and maltose) and five types of phosphate salts (Na₃PO₄, Na₄P₂O₇, Na₅P₃O₁₀, K₃PO₄, and K₄P₂O₇) were employed, and the thermal properties of various sugar-phosphate aqueous systems were investigated using differential scanning calorimetry. The T _g′ of glucose increased with increasing sodium phosphates up to a certain phosphate ratio, decreasing thereafter. The maximum T _g′ value was slightly higher in the order of Na₃PO₄ > Na₄P₂O₇ ≥ Na₅P₃O₁₀. Maltose-sodium phosphate also showed a similar trend as glucose-sodium phosphate samples. However, the degree of T _g′-rise of maltose systems was much less than that of glucose. It is thought that the T _g′ elevated by the molecular interaction between sugar and phosphate ions will be reduced by hydrated sodium ions. In comparisons between potassium phosphate and sodium phosphate, it was found that sugar-potassium phosphates showed the lower maximum T _g′ at a lower phosphate ratio than sugar-sodium phosphates. In addition, the T _g′ of potassium phosphates dropped sharply in comparison with sodium phosphates at the high phosphate ratio. These results suggest that potassium phosphates are lower T _g′ than sodium phosphates, and that potassium ion plays a better plasticizer than sodium ion. A certain amount of sodium phosphates (Na₃PO₄ and Na₄P₂O₇) caused devitrification. Potassium phosphates, however, did not show devitrification which can be explained by the fact that potassium ion can be dynamically restricted by sugar.     

AMPHOTERIC COLLOIDS : I. CHEMICAL INFLUENCE OF THE HYDROGEN ION CONCENTRATION.

1. It has been shown in this paper that while non-ionized gelatin may exist in gelatin solutions on both sides of the isoelectric point (which lies for gelatin at a hydrogen ion concentration of C_H = 2.10^–5 or pH = 4.7), gelatin, when it ionizes, can only exist as an anion on the less acid side of its isoelectric point (pH > 4.7), as a cation only on the more acid side of its isoelectric point (pH < 4.7). At the isoelectric point gelatin can dissociate practically neither as anion nor as cation. 2. When gelatin has been transformed into sodium gelatinate by treating it for some time with M/32 NaOH, and when it is subsequently treated with HCl, the gelatin shows on the more acid side of the isoelectric point effects of the acid treatment only; while the effects of the alkali treatment disappear completely, showing that the negative gelatin ions formed by the previous treatment with alkali can no longer exist in a solution with a pH < 4.7. When gelatin is first treated with acid and afterwards with alkali on the alkaline side of the isoelectric point only the effects of the alkali treatment are noticeable. 3. On the acid side of the isoelectric point amphoteric electrolytes can only combine with the anions of neutral salts, on the less acid side of their isoelectric point only with cations; and at the isoelectric point neither with the anion nor cation of a neutral salt. This harmonizes with the statement made in the first paragraph, and the experimental results on the effect of neutral salts on gelatin published in the writer''s previous papers. 4. The reason for this influence of the hydrogen ion concentration on the stability of the two forms of ionization possible for an amphoteric electrolyte is at present unknown. We might think of the possibility of changes in the configuration or constitution of the gelatin molecule whereby ionized gelatin can exist only as an anion on the alkaline side and as a cation on the acid side of its isoelectric point. 5. The literature of colloid chemistry contains numerous statements which if true would mean that the anions of neutral salts act on gelatin on the alkaline side of the isoelectric point, e.g. the alleged effect of the Hofmeister series of anions on the swelling and osmotic pressure of common gelatin in neutral solutions, and the statement that both ions of a neutral salt influence a protein simultaneously. The writer has shown in previous publications that these statements are contrary to fact and based on erroneous methods of work. Our present paper shows that these claims of colloid chemists are also theoretically impossible. 6. In addition to other physical properties the conductivity of gelatin previously treated with acids has been investigated and plotted, and it was found that this conductivity is a minimum in the region of the isoelectric point, thus confirming the conclusion that gelatin can apparently not exist in ionized condition at that point. The conductivity rises on either side of the isoelectric point, but not symmetrically for reasons given in the paper. It is shown that the curves for osmotic pressure, viscosity, swelling, and alcohol number run parallel to the curve of the conductivity of gelatin when the gelatin has been treated with acid, supporting the view that these physical properties are in this case mainly or exclusively a function of the degree of ionization of the gelatin or gelatin salt formed. It is pointed out, however, that certain constitutional factors, e.g. the valency of the ion in combination with the gelatin, may alter the physical properties of the gelatin (osmotic pressure, etc.) without apparently altering its conductivity. This point is still under investigation and will be further discussed in a following publication. 7. It is shown that the isoelectric point of an amphoteric electrolyte is not only a point where the physical properties of an ampholyte experience a sharp drop and become a minimum, but that it is also a turning point for the mode of chemical reactions of the ampholyte. It may turn out that this chemical influence of the isoelectric point upon life phenomena overshadows its physical influence. 8. These experiments suggest that the theory of amphoteric colloids is in its general features identical with the theory of inorganic hydroxides (e.g. aluminum hydroxide), whose behavior is adequately understood on the basis of the laws of general chemistry.     

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.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valence

There have been many reports suggesting the involvement of reactive oxygen species (ROS), including superoxide anion (O₂^.–), in salt stress. Herein, direct evidence that treatments of cell suspension culture of tobacco (Nicotiana tabacum L.; cell line, BY‐2) with various salts of trivalent, divalent and monovalent metals stimulate the immediate production of O₂^.– is reported. Among the salts tested, LaCl₃ and GdCl₃ induced the greatest responses in O₂^.– production, whereas CaCl₂ and MgCl₂ showed only moderate effects; salts of monovalent metals such as KCl and NaCl induced much lower responses, indicating that there is a strong relationship between the valence of metals and the level of O₂^.– production. As the valence of the added metals increased from monovalent to divalent and trivalent, the concentrations required for maximal responses were lowered. Although O₂^.– production by NaCl and KCl required high concentrations associated with hyperosmolarity, the O₂^.– generation induced by NaCl and KCl was significantly greater than that induced simply by hyperosmolarity. Since an NADPH oxidase inhibitor, diphenyleneiodonium chloride, showed a strong inhibitory effect on the trivalent and divalent cation‐induced generation of O₂^.–, it is likely that cation treatments activate the O₂^.–‐generating activity of NADPH oxidase.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

Impact of inorganic salts on degradation of bisphenol A and diclofenac by crude extracellular enzyme from Pleurotus ostreatus

This study investigated the influence of inorganic salts on enzymatic activity and the removal of trace organic contaminants (TrOCs) by crude laccase from the white-rot fungus Pleurotus ostreatus. A systematic analysis of 15 cations and anions from common inorganic salts was presented. Laccase activity was not inhibited by monovalent cations (i.e. Na⁺, NH₄⁺, K⁺), while the presence of divalent and trivalent cations showed variable impact – from negligible to complete inhibition – of both laccase activity and its TrOC removal performance. Of interest was the observation of discrepancy between residual laccase activity and TrOC removal in the presence of some ions. Mg²⁺ had negligible impact on residual laccase activity but significant impact on TrOC removal. Conversely, F^? showed greater impact on residual laccase activity than on TrOC removal. This observation indicated different impacts of the interfering ions on the interaction between laccase and TrOCs as compared to that between laccase and the reagent used to measure its activity, implicating that residual laccase activity may not always be an accurate indicator of TrOC removal. The degree of impact of halides was in the order of F^??>?I^? >?Br^??>?Cl^?. Particularly, the tolerance of the tested laccase to Cl^? has important implications for a range of industrial applications.     

Salt contribution to the flexibility of single‐stranded nucleic acid of finite length

Nucleic acids are negatively charged macromolecules and their structure properties are strongly coupled to metal ions in solutions. In this article, the salt effects on the flexibility of single‐stranded (ss) nucleic acid chain ranging from 12 to 120 nucleotides are investigated systematically by the coarse‐grained Monte Carlo simulations where the salt ions are considered explicitly and the ss chain is modeled with the virtual‐bond structural model. Our calculations show that, the increase of ion concentration causes the structural collapse of ss chain and multivalent ions are much more efficient in causing such collapse, and both trivalent/small divalent ions can induce more compact state than a random relaxation state. We found that monovalent, divalent, and trivalent ions can all overcharge ss chain, and the dominating source for such overcharging changes from ion‐exclusion‐volume effect to ion Coulomb correlations. In addition, the predicted Na⁺ and Mg²⁺‐dependent persistence length l_p’s of ss nucleic acid are in accordance with the available experimental data, and through systematic calculations, we obtained the empirical formulas for l_p as a function of [Na⁺], [Mg²⁺] and chain length. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 370–381, 2013.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Effect of Pressure-Induced Changes in the Ionization Equilibria of Buffers on Inactivation of Escherichia coli and Staphylococcus aureus by High Hydrostatic Pressure

Survival rates of Escherichia coli and Staphylococcus aureus after high-pressure treatment in buffers that had large or small reaction volumes (ΔV°), and which therefore underwent large or small changes in pH under pressure, were compared. At a low buffer concentration of 0.005 M, survival was, as expected, better in MOPS (morpholinepropanesulfonic acid), HEPES, and Tris, whose ΔV° values are approximately 5.0 to 7.0 cm³ mol⁻¹, than in phosphate or dimethyl glutarate (DMG), whose ΔV° values are about −25 cm³ mol⁻¹. However, at a concentration of 0.1 M, survival was unexpectedly better in phosphate and DMG than in MOPS, HEPES, or Tris. This was because the baroprotective effect of phosphate and DMG increased much more rapidly with increasing concentration than it did with MOPS, HEPES, or Tris. Further comparisons of survival in solutions of salts expected to cause large electrostriction effects (Na₂SO₄ and CaCl₂) and those causing lower electrostriction (NaCl and KCl) were made. The salts with divalent ions were protective at much lower concentrations than salts with monovalent ions. Buffers and salts both protected against transient membrane disruption in E. coli, but the molar concentrations necessary for membrane protection were much lower for phosphate and Na₂SO₄ than for HEPES and NaCl. Possible protective mechanisms discussed include effects of electrolytes on water compressibility and kosmotropic and specific ion effects. The results of this systematic study will be of considerable practical significance in studies of pressure inactivation of microbes under defined conditions but also raise important fundamental questions regarding the mechanisms of baroprotection by ionic solutes.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Adenosine triphosphatase from soybean callus and root cells

The ATPase activity of a membrane fraction from soybean (Glycine max L.) root and callus cells, presumed to be enriched in plasma membrane, has been characterized with respect to ion stimulation, pH requirement, and nucleotide specificity. The enzyme from both sources was activated by divalent cations (Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺ > Sr²⁺) and further stimulated by monovalent salts. Preparations from root cells were stimulated by monovalent ions according to the sequence: K⁺ > Rb⁺ > Choline⁺ > Na⁺ > Li⁺ > NH₄⁺ > Cs⁺ > tris⁺. Membrane preparations from callus cells showed similar stimulatory patterns except for a slight preference for Na⁺ over K⁺. No synergism between K⁺ and Na⁺ was found with preparations from either cell source.     

Effect of Ethanol on the Sodium and Potassium Conductances of the Squid Axon Membrane

The effects of ethanol on squid giant axons were studied by means of the sucrose-gap technique. The membrane action potential height is moderately reduced and the duration sometimes shortened by ethanol in sea water. Voltage clamp experiments showed that ethanol in sea water reduced the maximum membrane conductances for sodium (g'_Na) and potassium (g'_K). In experiments with multiple application of ethyl alcohol to the same spot of membrane, a reduction of g'_Na to 82 per cent and of g'_K to 80 per cent of their value in sea water was brought about by 3 per cent ethanol (by volume) while 6 per cent caused a decrease of g'_Na to 59 per cent and of g'_K to 69 per cent. Ethanol has no significant effect on the steady-state inactivation of g_Na (as a function of conditioning membrane potential) or on such kinetic parameters as τ_h or the time course of turning on gi g_Na and g_K. It is concluded that ethanol mainly reduces g_Na and g_K in the Hodgkin-Huxley terminology.