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.     

DONNAN EQUILIBRIUM AND THE PHYSICAL PROPERTIES OF PROTEINS : II. OSMOTIC PRESSURE.

1. It had been shown in previous publications that the osmotic pressure of a 1 per cent solution of a protein-acid salt varies in a characteristic way with the hydrogen ion concentration of the solution, the osmotic pressure having a minimum at the isoelectric point, rising steeply with a decrease in pH until a maximum is reached at pH of 3.4 or 3.5 (in the case of gelatin and crystalline egg albumin), this maximum being followed by a steep drop in the osmotic pressure with a further decrease in the pH of the gelatin or albumin solution. In this paper it is shown that (aside from two minor discrepancies) we can calculate this effect of the pH on the osmotic pressure of a protein-acid salt by assuming that the pH effect is due to that unequal distribution of crystalloidal ions (in particular free acid) on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 2. It had been shown in preceding papers that only the valency but not the nature of the ion (aside from its valency) with which a protein is in combination has any effect upon the osmotic pressure of the solution of the protein; and that the osmotic pressure of a gelatin-acid salt with a monovalent anion (e.g. Cl, NO₃, acetate, H₂PO₄, HC₂O₄, etc.) is about twice or perhaps a trifle more than twice as high as the osmotic pressure of gelatin sulfate where the anion is bivalent; assuming that the pH and gelatin concentrations of all the solutions are the same. It is shown in this paper that we can calculate with a fair degree of accuracy this valency effect on the assumption that it is due to the influence of the valency of the anion of a gelatin-acid salt on that relative distribution of the free acid on both sides of the membrane which Donnan''s theory of membrane equilibrium demands. 3. The curves of the observed values of the osmotic pressure show two constant minor deviations from the curves of the calculated osmotic pressure. One of these deviations consists in the fact that the values of the ascending branch of the calculated curves are lower than the corresponding values in the curves for the observed osmotic pressure, and the other deviation consists in the fact that the drop in the curves of calculated values occurs at a lower pH than the drop in the curves of the observed values.     

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.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. II

1. Our results show clearly that the Hofmeister series is not the correct expression of the relative effect of ions on the swelling of gelatin, and that it is not true that chlorides, bromides, and nitrates have "hydrating," and acetates, tartrates, citrates, and phosphates "dehydrating," effects. If the pH of the gelatin is taken into considertion, it is found that for the same pH the effect on swelling is the same for gelatin chloride, nitrate, trichloracetate, tartrate, succinate, oxalate, citrate, and phosphate, while the swelling is considerably less for gelatin sulfate. This is exactly what we should expect on the basis of the combining ratios of the corresponding acids with gelatin since the weak dibasic and tribasic acids combine with gelatin in molecular proportions while the strong dibasic acid H₂SO₄ combines with gelatin in equivalent proportions. In the case of the weak dibasic acids he anion in combination with gelatin is therefore monovalent and in the case of the strong H₂SO₄ it is bivalent. Hence it is only the valency and not the nature of the ion in combination with gelatin which affects the degree of swelling. 2. This is corroborated in the experiments with alkalies which show that LiOH, NaOH, KOH, and NH₄OH cause the same degree of swelling at the same pH of the gelatin solution and that this swelling is considerably higher than that caused by Ca(OH)₂ and Ba(OH)₂ for the same pH. This agrees with the results of the titration experiments which prove that Ca(OH)₂ and Ba(OH)₂ combine with gelatin in equivalent proportions and that hence the cation in combination with the gelatin salt with these two latter bases is bivalent. 3. The fact that proteins combine with acids and alkalies on the basis of the forces of primary valency is therefore not only in full agreement with the influence of ions on the physical properties of proteins but allows us to predict this influence qualitatively and quantitatively. 4. What has been stated in regard to the influence of ions on the swelling of the different gelatin salts is also true in regard to the influence of ions on the relative solubility of gelatin in alcohol-water mixtures. 5. Conductivity measurements of solutions of gelatin salts do not support the theory that the drop in the curves for swelling, osmotic pressure, or viscosity, which occurs at a pH 3.3 or a little less, is due to a drop in the concentration of ionized protein in the solution; nor do they suggest that the difference between the physical properties of gelatin sulfate and gelatin chloride is due to differences in the degree of ionization of these two salts.     

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.     

VALENCY RULE AND ALLEGED HOFMEISTER SERIES IN THE COLLOIDAL BEHAVIOR OF PROTEINS : I. THE ACTION OF ACIDS.

1. The action of a number of acids on four properties of gelatin (membrane potentials, osmotic pressure, swelling, and viscosity) was studied. The acids used can be divided into three groups; first, monobasic acids (HCl, HBr, HI, HNO₃, acetic, propionic, and lactic acids); second, strong dibasic acids (H₂SO₄ and sulfosalicylic acid) which dissociate as dibasic acids in the range of pH between 4.7 and 2.5; and third, weak dibasic and tribasic acids (succinic, tartaric, citric) which dissociate as monobasic acids at pH 3.0 or below and dissociate increasingly as dibasic acids, according to their strength, with pH increasing above 3.0. 2. If the influence of these acids on the four above mentioned properties of gelatin is plotted as ordinates over the pH of the gelatin solution or gelatin gel as abscissæ, it is found that all the acids have the same effect where the anion is monovalent; this is true for the seven monobasic acids at all pH and for the weak dibasic and tribasic acids at pH below 3.0. The two strong dibasic acids (the anion of which is divalent in the whole range of pH of these experiments) have a much smaller effect than the acids with monovalent anion. The weak dibasic and tribasic acids act, at pH above 3.0, like acids the anion of which is chiefly monovalent but which contain also divalent anions increasing with pH and with the strength of the acid. 3. These experiments prove that only the valency but not the other properties of the anion of an acid influences the four properties of gelatin mentioned, thus absolutely contradicting the Hofmeister anion series in this case which were due to the failure of the earlier experimenters to measure properly the pH of their protein solutions or gels and to compare the effects of acids at the same pH of the protein solution or protein gel after equilibrium was established. 4. It is shown that the validity of the valency rule and the non-validity of the Hofmeister anion series for the four properties of proteins mentioned are consequences of the fact that the influence of acids on the membrane potentials, osmotic pressure, swelling, and viscosity of gelatin is due to the Donnan equilibrium between protein solutions or gels and the surrounding aqueous solution. This equilibrium depends only on the valency but not on any other property of the anion of an acid. 5. That the valency rule is determined by the Donnan equilibrium is strikingly illustrated by the ratio of the membrane potentials for divalent and monovalent anions of acids. Loeb has shown that the Donnan equilibrium demands that this ratio should be 0.66 and the actual measurements agree with this postulate of the theory within the limits of accuracy of the measurements. 6. The valency rule can be expected to hold for only such properties of proteins as depend upon the Donnan equilibrium. Properties of proteins not depending on the Donnan equilibrium may be affected not only by the valency but also by the chemical nature of the anion of an acid.     

ELECTRIFICATION OF WATER AND OSMOTIC PRESSURE

1. Amphoteric electrolytes form salts with both acids and alkalies. It is shown for two amphoteric electrolytes, Al(OH)₃ and gelatin, that in the presence of an acid salt water diffuses through a collodion membrane into a solution of these substances as if its particles were negatively charged, while water diffuses into solutions of these electrolytes, when they exist as monovalent or bivalent metal salts, as if the particles of water were positively charged. The turning point for the sign of the electrification of water seems to be near or to coincide with the isoelectric point of these two ampholytes which is a hydrogen ion concentration of about 2 x 10^–5 N for gelatin and about 10^–7 for Al(OH)₃. 2. In conformity with the rules given in a preceding paper the apparently positively charged water diffuses with less rapidity through a collodion membrane into a solution of Ca and Ba gelatinate than into a solution of Li, Na, K, or NH₄ gelatinate of the same concentration of gelatin and of hydrogen ions. Apparently negatively charged water diffuses also with less rapidity through a collodion membrane into a solution of gelatin sulfate than into a solution of gelatin chloride or nitrate of the same concentration of gelatin and of hydrogen ions. 3. If we define osmotic pressure as that additional pressure upon the solution required to cause as many molecules of water to diffuse from solution to the pure water as diffuse simultaneously in the opposite direction through the membrane, it follows that the osmotic pressure cannot depend only on the concentration of the solute but must depend also on the electrostatic effects of the ions present and that the influence of ions on the osmotic pressure must be the same as that on the initial velocity of diffusion. This assumption was put to a test in experiments with gelatin salts for which a collodion membrane is strictly semipermeable and the tests confirmed the expectation.     

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.     

AMPHOTERIC COLLOIDS : V. THE INFLUENCE OF THE VALENCY OF ANIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. When we plot the values of osmotic pressure, swelling, and viscosity of gelatin solutions as ordinates over the pH as abscissæ, practically identical curves are obtained for the effect of monobasic acids (HCl, HBr, HNO₃, and acetic acid) on these properties. 2. The curves obtained for the effect of H₂SO₄ on gelatin are much lower than those obtained for the effect of monobasic acids, the ratio of maximal osmotic pressures of a 1 per cent solution of gelatin sulfate and gelatin bromide being about 3:8. The same ratio had been found for the ratio of maximal osmotic pressures of calcium and sodium gelatinate. 3. The curves representing the influence of other dibasic and tribasic acids, viz. oxalic, tartaric, succinic, citric, and phosphoric, upon gelatin are almost identical with those representing the effect of monobasic acids. 4. The facts mentioned under (2) and (3) permit us to decide between a purely chemical and a colloidal explanation of the influence of acids on the physical properties of gelatin. In the former case we should be able to prove, first, that twice as many molecules of HBr as of H₂SO₄ combine with a given mass of gelatin; and, second, that the same number of molecules of phosphoric, citric, oxalic, tartaric, and succinic acids as of HNO₃ or HCl combine with the same mass of gelatin. It is shown in the present paper that this is actually the case. 5. It is shown that gelatin sulfate and gelatin bromide solutions of the same pH have practically the same conductivity. This disproves the assumption of colloid chemists that the difference in the effect of bromides and sulfates on the physical properties of gelatin is due to a different ionizing and hydratating effect of the two acids upon the protein molecule.     

AMPHOTERIC COLLOIDS : IV. THE INFLUENCE OF THE VALENCY OF CATIONS UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. A method is given by which the amount of equivalents of metal in combination with 1 gm. of a 1 per cent gelatin solution previously treated with an alkali can be ascertained when the excess of alkali is washed away and the pH is determined. The curves of metal equivalent in combination with 1 gm. of gelatin previously treated with different concentrations of LiOH, NaOH, KOH, NH₄OH, Ca(OH)₂, and Ba(OH)₂ were ascertained and plotted as ordinates, with the pH of the solution as abscissæ, and were found to be identical. This proves that twice as many univalent as bivalent cations combine with the same mass of gelatin, as was to be expected. 2. The osmotic pressure of 1 per cent solutions of metal gelatinates with univalent and bivalent cation was measured. The curves for the osmotic pressure of 1 per cent solution of gelatin salts of Li, Na, K, and NH₄ were found to be identical when plotted for pH as abscissæ, tending towards the same maximum of a pressure of about 325 mm. of the gelatin solution (for pH about 7.9). The corresponding curves for Ca and Ba gelatinate were also found to be identical but different from the preceding ones, tending towards a maximum pressure of about 125 mm. for pH about 7.0 or above. The ratio of maxi mal osmotic pressure for the two groups of gelatin salts is therefore about as 1:3 after the necessary corrections have been made. 3. When the conductivities of these solutions are plotted as ordinates against the pH as abscissæ, the curves for the conductivities of Li, Na, Ca, and Ba gelatinate are almost identical (for the same pH), while the curves for the conductivities of K and NH₄ gelatinate are only little higher. 4. The curves for the viscosity and swelling of Ba (or Ca) and Na gelatinate are approximately parallel to those for osmotic pressure. 5. The practical identity or close proximity of the conductivities of metal gelatinates with univalent and bivalent metal excludes the possibility that the differences observed in the osmotic pressure, viscosity, and swelling between metal gelatinates with univalent and bivalent metal are determined by differences in the degree of ionization (and a possible hydratation of the protein ions). 6. Another, as yet tentative, explanation is suggested.     

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.     

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van''t Hoff''s law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H₂O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin.     

AMPHOTERIC COLLOIDS : III. CHEMICAL BASIS OF THE INFLUENCE OF ACID UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. The method of removing the excess of hydrobromic acid after it has had a chance to react chemically with gelatin has permitted us to measure the amount of Br in combination with the gelatin. It is shown that the curves representing the amount of bromine bound by the gelatin are approximately parallel with the curves for the osmotic pressure, the viscosity, and swelling of the gelatin solution. This proves that the curves for osmotic pressure are an unequivocal function of the number of gelatin bromide molecules formed under the influence of the acid. The cc. of 0.01 N Br in combination with 0.25 gm, of gelatin we call the bromine number. 2. The explanation of this influence of the acid on the physical properties of gelatin is based on the fact that gelatin is an amphoteric electrolyte, which at its isoelectric point is but sparingly soluble in water, while its transformation into a salt with a univalent anion like gelatin Br makes it soluble. The curve for the bromine number thus becomes at the same time the numerical expression for the number of gelatin molecules rendered soluble, and hence the curve for osmotic pressure must of necessity be parallel to the curve for the bromine number. 3. Volumetric analysis shows that gelatin treated previously with HBr is free from Br at the isoelectric point as well as on the more alkaline side from the isoelectric point (pH ≧ 4.7) of gelatin. This is in harmony with the fact that gelatin (like any other amphoteric electrolyte) can dissociate on the alkaline side of its isoelectric point only as an anion. On the more acid side from the isoelectric point gelatin is found to be in combination with Br and the Br number rises with the pH. 4. When we titrate gelatin, treated previously with HBr but possessing a pH = 4,7, with NaOH we find that 25 cc. of a 1 per cent solution of isoelectric gelatin require about 5.25 to 5.5 cc. of 0.01 N NaOH for neutralization (with phenolphthalein as an indicator). This value which was found invariably is therefore a constant which we designate as "NaOH (isoelectric)." When we titrate 0.25 gm. of gelatin previously treated with HBr but possessing a pH < 4.7 more than 5.5 cc. of 0.01 N NaOH are required for neutralization. We will designate this value of NaOH as "(NaOH)_n," where n represents the value of pH. If we designate the bromine number for the same pH as "Br_n" then we can show that the following equation is generally true: (NaOH)_n = NaOH (isoelectric) + Br_n. In other words, titration with NaOH of gelatin (previously treated with HBr) and being on the acid side of its isoelectric point results in the neutralization of the pure gelatin (NaOH isoelectric) with NaOH and besides in the neutralization of the HBr in combination with the gelatin. This HBr is set free as soon as through the addition of the NaOH the pH of the gelatin solution becomes equal to 4.7. 5. A comparison between the pH values and the bromine numbers found shows that over 90 per cent of the bromine or HBr found was in our experiments in combination with the gelatin.     

THE SWELLING AND OSMOTIC PRESSURE OF GELATIN IN SALT SOLUTIONS

1. The swelling and the osmotic pressure of gelatin at pH 4.7 have been measured in the presence of a number of salts. 2. The effect of the salts on the swelling is closely paralleled by the effect on the osmotic pressure, and the bulk modulus of the gelatin particles calculated from these figures is constant up to an increase in volume of about 800 per cent. As soon as any of the salts increase the swelling beyond this point, the bulk. modulus decreases. This is interpreted as showing that the elastic limit has been exceeded. 3. Gelatin swollen in acid returns to its original volume after removal of the acid, while gelatin swollen in salt solution does not do so. This is the expected result if, as stated above, the elastic limit had been exceeded in the salt solution. 4. The modulus of elasticity of gelatin swollen in salt solutions varies in the same way as the bulk modulus calculated from the osmotic pressure and the swelling. 5. The increase in osmotic pressure caused by the salt is reversible on removal of the salt. 6. The observed osmotic pressure is much greater than the osmotic pressure calculated from the Donnan equilibrium except in the case of AlCl₃, where the calculated and observed pressures agree quite closely. 7. The increase in swelling in salt solutions is due to an increase in osmotic pressure. This increase is probably due to a change in the osmotic pressure of the gelatin itself rather than to a difference in ion concentration.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS : III. THE ACTION OF SALTS IN LOW CONCENTRATION.

1. Ions with the opposite sign of charge as that of a protein ion diminish the swelling, osmotic pressure, and viscosity of the protein. Ions with the same sign of charge as the protein ion (with the exception of H and OH ions) seem to have no effect on these properties as long as the concentrations of electrolytes used are not too high. 2. The relative depressing effect of different ions on the physical properties of proteins is a function only of the valency and sign of charge of the ion, ions of the same sign of charge and the same valency having practically the same depressing effect on gelatin solutions of the same pH while the depressing effect increases rapidly with an increase in the valency of the ion. 3. The Hofmeister series of ions are the result of an error due to the failure to notice the influence of the addition of a salt upon the hydrogen ion concentration of the protein solution. As a consequence of this failure, effects caused by a variation in the hydrogen ion concentration of the solution were erroneously attributed to differences in the nature of the ions of the salts used. 4. It is not safe to draw conclusions concerning specific effects of ions on the swelling, osmotic pressure, or viscosity of gelatin when the concentration of electrolytes in the solution exceeds M/16, since at that concentration the values of these properties are near the minimum characteristic of the isoelectric point.     

CASEIN VISCOSITY STUDIES

1. Viscosity and pH curves of casein dissolved in NaOH, KOH, LiOH, and NH₄OH are shown and it is found that a maximum viscosity occurs at about the same pH point with each alkali; i.e., 9.1 to 9.25. The magnitude of the viscosity is largest in ammonia solutions. 2. The maximum viscosity occurs in 8 to 10 per cent solutions of casein in alkalies when about 98 x 10^–5 gram equivalents of base are combined with 1 gram of casein. 3. A maximum viscosity occurs in the same region (pH 9.1 to 9.25) when casein is dissolved in Na₂CO₃, Na₃AsO₄, Na₂SO₃, NaF, and Na₂PO₃. 4. The maximum viscosity obtained with borax solutions of casein occurs at 8.15 to 8.2 pH. It is suggested that casein acts like mannitol, glycerol, etc., in increasing the dissociation of boric acid. 5. The flattening of the viscosity curves of casein solutions, following the decline from maximum, is shown to be due to alkaline hydrolysis whence casein no longer exists as such but is cleaved into a major protein containing no phosphorus or sulfur and less nitrogen. This cleavage commences at pH 10.0 to 10.5. 6. When casein is prepared from solutions that have been subjected to high temperatures (60°C. and above) or has otherwise been heated during its preparation, it yields solutions in alkalies of high viscosity.     

SYNERESIS AND SWELLING OF GELATIN

1. When solid blocks of isoelectric gelatin are placed in cold distilled water or dilute buffer of pH 4.7, only those of a gelatin content of more than 10 per cent swell, while those of a lower gelatin content not only do not swell but actually lose water. 2. The final quantity of water lost by blocks of dilute gelatin is the same whether the block is immersed in a large volume of water or whether syneresis has been initiated in the gel through mechanical forces such as shaking, pressure, etc., even in the absence of any outside liquid, thus showing that syneresis is identical with the process of negative swelling of dilute gels when placed in cold water, and may be used as a convenient term for it. 3. Acid- or alkali-containing gels give rise to greater syneresis than isoelectric gels, after the acid or alkali has been removed by dialysis. 4. Salt-containing gels show greater syneresis than salt-free gels of the same pH, after the salt has been washed away. 5. The acid and alkali and also the salt effect on syneresis of gels disappears at a gelatin concentration above 8 per cent. 6. The striking similarity in the behavior of gels with respect to syneresis and of gelatin solutions with respect to viscosity suggests the probability that both are due to the same mechanism, namely the mechanism of hydration of the micellæ in gelatin by means of osmosis as brought about either by diffusible ions, as in the presence of acid or alkali, or by the soluble gelatin present in the micellæ. The greater the pressures that caused swelling of the micellæ while the gelatin was in the sol state, the greater is the loss of water from the gels when the pressures are removed. 7. A quantitative study of the loss of water by dilute gels of various gelatin content shows that the same laws which have been found by Northrop to hold for the swelling of gels of high concentrations apply also to the process of losing water by dilute gels, i.e. to the process of syneresis. The general behavior is well represented by the equations: See PDF for Equation and See PDF for Equation where P ₁ = osmotic pressure of the soluble gelatin in the gel, P ₂ = stress on the micellæ in the gelatin solution before setting, K_e = bulk modulus of elasticity, V_o = volume of water per gram of dry gelatin at setting and V_e = volume of water per gram of gelatin at equilibrium.     

IONIZING INFLUENCE OF SALTS WITH TRIVALENT AND TETRAVALENT IONS ON CRYSTALLINE EGG ALBUMIN AT THE ISOELECTRIC POINT

1. While crystalline egg albumin is highly soluble in water at low temperature at the pH of its isoelectric point, it is coagulated by heating. It has long been known that this coagulation can be prevented by adding either acid or alkali, whereby the protein is ionized. 2. It is shown in this paper that salts with trivalent or tetravalent ions, e.g. LaCl₃ or Na₄Fe(CN)₆, are also able to prevent the heat coagulation of albumin at the isoelectric point (i.e. pH 4.8), while salts with a divalent ion, e.g. CaCl₂, BaCl₄, Na₂SO₄, or salts like NaCl, have no such effect. 3. This is in harmony with the fact shown in a preceding paper that salts with trivalent or tetravalent ions can cause the ionization of proteins at its isoelectric point and thus give rise to a membrane potential between micellæ of isoelectric protein and surrounding aqueous solution, while the above mentioned salts with divalent and monovalent ions have apparently no such effect.     

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.     

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.