THE SWELLING OF ISOELECTRIC GELATIN IN WATER : II. KINETICS.

It has been assumed that gelatin consists of a network of an insoluble material enclosing a solution of a more soluble material. The swelling of gelatin is therefore primarily an osmotic phenomena in that the water tends to diffuse in owing to the osmotic pressure of the soluble material. This osmotic pressure is opposed by the elasticity of the insoluble constituent, and equilibrium results when these two pressures are equal. The rate of the entrance of water should then obey Poiseuille''s law, provided the variable terms are expressed as functions of the volume. Equations have been derived in this way which agree quite well with the experimental curves and which predict the proper relation between the size and shape of the block and the rate of swelling. They lead to a value for the rate of flow of water through gelatin which has been checked by direct measurement. The mechanism assumed predicts that at a higher temperature and under conditions such that the water has to pass through collodion before reaching the gelatin, the experiment should follow the same course as that of osmosis discussed previously. This was also found to be the case. The slow secondary increase in swelling is ascribed to fatigue of the elastic properties of the gelatin. The rate of this secondary swelling should therefore be independent of the size of the block, in contrast to the rate of primary swelling which is inversely proportional to the size. It can further be shown that this secondary swelling should be proportional to the square root of the time, and also that with large blocks at higher temperatures the entire swelling should be of this secondary type. These predictions have also been found to be true.     

THE SWELLING PRESSURE OF GELATIN AND THE MECHANISM OF SWELLING IN WATER AND NEUTRAL SALT SOLUTIONS

1. A method is described for measuring the swelling pressure of solid gelatin. 2. It was found that this pressure increases rapidly between 15° and 37°C., and that the percentage change is nearly independent of the concentration of gelatin. 3. It is suggested that this pressure is due to the osmotic pressure of a soluble constituent of the gelatin held in the network of insoluble fibers, and that gelatin probably consists of a mixture of at least two substances or groups of substances, one of which is soluble in cold water, does not form a gel, and has a low viscosity and a high osmotic pressure. The second is insoluble in cold water, forms a gel in very low concentration, and swells much less than ordinary gelatin. 4. Two fractions, having approximately the above properties, were isolated from gelatin by alcohol precipitation at different temperatures. 5. Increasing the temperature and adding neutral salts greatly increase the pressure of the insoluble fraction and have little effect on that of the soluble fraction. 6. Adding increasing amounts of the soluble fraction to the insoluble one results in greater and greater swelling. 7. These results are considered as evidence for the idea that the swelling of gelatin in water or salt solutions is an osmotic phenomenon, and that gelatin consists of a network of an insoluble substance enclosing a solution of a soluble constituent.     

THE FLOCCULATION OF GELATIN AT THE ISOELECTRIC POINT

The results of this investigation show that a gelatin solution consists of a considerable number of constituents. At a particular temperature, certain gelatin constituents tend to aggregate and to flocculate from solution. When these particular gelatin constituents have completely flocculated, no further change occurs in the system and an apparent equilibrium exists. This is not a dynamic equilibrium between the gelatin flocculate as a whole and the gelatin remaining in the solution but a steady state determined for that system by the temperature. It is also shown that gelatin can be separated into fractions in which the gelatin constituents are more nearly uniform and tend to flocculate over a much narrower temperature range. It should be possible to obtain a number of fractions in which all of the gelatin would flocculate at a definite temperature. The aggregation of the various gelatin constituents is presumably due to loss of thermal energy, and the temperature at which this occurs must be some function of the mass of the constituent. It is natural to assume, then, that the constituents which flocculate at a given temperature are larger than those which remain in solution at that temperature. Recently, Krishnamurti and Svedberg (1930) have obtained evidence with the ultra-centrifuge that the constituents of a gelatin solution are heterogeneous as to mass, even at a pH value at which there is no tendency toward aggregation. There is much reason to suppose that the gelatin constituents do not differ very greatly chemically since different fractions have the same refractive index and the same isoelectric point. The data as a whole are best explained by considering the gelatin constituents to be different degrees of association of the same or very similar molecular structural units. This is in agreement with Sheppard and Houck (1930), who consider that "the molecules of gelatin are fundamentally identical with those of collagen, the difference being only in the degree of association and orientation". Meyer and Mark (1928) have interpreted the x-ray data obtained from collagen as indicating that the micelles of the collagen fiber are built up of main valency chains of anhydro-amino acids. It may be supposed that during peptization of these fibers, the amino acid chains become separated, disorientated, and partially broken up, so producing the heterogeneous system which we know as gelatin. It is evident that the manner in which this breaking-up proceeds depends upon the chemical treatment previous to the peptization process and the gelatin produced from lime-treated collagen would be expected to differ from that from acid-treated collagen. From the results herein reported it seems evident that the technique of isoelectric flocculation of electrolyte-free gelatin offers a profitable method for the study of gelatin and an extended investigation along these lines should yield much valuable information concerning the nature of gelatin. It is possible that this method may also be extended to other hydrophilic colloids.     

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.     

THE SWELLING OF GELATIN AND THE VOLUME OF SURROUNDING SOLUTION

The swelling of isoelectric gelatin added to various volumes of acid of different concentration at 5°C. has been determined. The swelling is determined only by the concentration of the supernatant solution at equilibrium and is independent of the volume of acid. Similar experiments with unpurified gelatin show that in this case, owing to the presence of neutral salts the swelling is a function of the volume as well as the concentration of acid. Both results are predicted by the Procter-Wilson-Loeb theory of the swelling of gelatin.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID : II. NEW DETERMINATIONS OF THE ISOELECTRIC POINT AND COMBINING CAPACITY OF A PURIFIED GELATIN.

1. Cooper''s gelatin purified according to Northrop and Kunitz exhibited a minimum of osmotic pressure and a maximum of opacity at pH 5.05 ±0.05. The pH of solutions of this gelatin in water was also close to this value. It is inferred that such gelatin is isoelectric at this pH and not at pH 4.70. 2. Hydrogen electrode measurements with KCl-agar junctions were made with concentrated solutions of this gelatin in HCl up to 0.1 M. The combination curve calculated from these data is quite exactly horizontal between pH 2 and 1, indicating that 1 gm. of this gelatin can combine with a maximum of 9.35 x 10^–4 equivalents of H⁺. 3. Conductivity titrations of this gelatin with HCl gave an endpoint at 9.41 (±0.05) x 10^–4 equivalents of HCl per gram gelatin. 4. E.M.F. measurements of the cell without liquid junction, Ag, AgCl, HCl + gelatin, H₂, lead to the conclusion that this gelatin in 0.1 M HCl combines with a maximum of 9.4 x 10^–4 equivalents of H⁺ and 1.7 x 10^–4 equivalents of Cl^- per gram gelatin.     

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.     

THE SORPTION OF BACTERIOPHAGE BY LIVING AND DEAD SUSCEPTIBLE BACTERIA : I. EQUILIBRIUM CONDITIONS

The above data relating to the antistaphylococcus phage and single strain of S. aureus with which previous papers have been concerned (9, 10, 11, 12), bring out the following points. (a) For live, resting, susceptible B suspended in broth as well as for B in a P-B mixture during the logarithmic phases of B growth and P formation, P is distributed in a manner typical of numerous materials soluble in both phases of a two phase system, i.e., distribution proceeds in accordance with the equation C_b/C_a = K where C_b = extracellular P/ml. of broth and C_a = intracellular P/ml. of B. The combination is quantitatively reversible. (b) With heat-killed, susceptible B, P distribution is of the adsorptive type, expressible in the form of the adsorption isotherm equation a = kC ^1/n. The average value of 1/n is 0.80 in agreement with the results of von Angerer (2). Under ordinary conditions dead B take up much more P than do live B, the reaction proceeding to > 99 per cent completion. The combination of P with dead B is not demonstrably reversible and with high initial P/B ratios saturation of B with P is effected. Bacteria killed at 65°C., 80°C. and 100°C. show no differences in adsorptive ability. (c) The rates at which live, resting, susceptible B and heat-killed, susceptible B remove P from solution do not differ significantly. Velocity constants of the process calculated from See PDF for Equation agree satisfactorily. It is shown that the time interval consumed is concerned with an actual reaction between P and B and not with diffusion of P through the broth to B. (d) P determinations have been found to serve as satisfactory indicators for B growth in P-B mixtures where [B] is to be maintained at a constant level. Very small increments in [B] give rise to measurable increases in P by virtue of the fact that dP/dt is proportional to a power of the rate dB/dt (9). (e) Similarly [P] estimations will detect death of B cells in P-live B suspensions. Dead B take up large amounts of P irreversibly; such P cannot function in the titration and the result is a sharp drop in [P] of controls.     

THE ISOELECTRIC POINT OF A STANDARD GELATIN PREPARATION1

Two samples of a standard gelatin were studied, both prepared according to published specifications and washed free from diffusible electrolytes. The isoelectric point of this material was determined in four ways. 1. The pH values of solutions of gelatin in water approached the limit 4.86 ± 0.01 as the concentration of gelatin was increased. 2. The pH values of acetate buffers were unchanged by the addition of gelatin only at pH 4.85 ± 0.01. This gives the isoionic point of Sørensen, which is the isoelectric point with respect only to hydrogen and hydroxyl ions. 3. Gels of this gelatin made up in dilute HCl or NaOH, or in dilute acetate buffers, exhibited maximum turbidity at pH 4.85 ± 0.03. 4. Very dilute suspensions of collodion particles in 0.1 per cent gelatin solutions made up in acetate buffers showed zero velocity in cataphoresis experiments only at pH 4.80 ± 0.01. No evidence was found for the assumption that gelatin has two isoelectric points at widely separated pH values. It is concluded that the isoelectric point of this standard gelatin is not far from pH 4.85.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : I. THE SOLUBILITY OF CERTAIN PROTEINS AT THEIR ISOELECTRIC POINTS.

1. Two proteins of the globulin type, serum globulin and tuberin, and the protein of milk, casein, have been purified (a) of the other proteins and (b) of the inorganic electrolytes with which they exist in nature. The methods that were employed are described. 2. All three proteins were found to be only very slightly soluble in water in the pure uncombined state. The solubility of each was accurately measured at 25.0° ± 0.1°C. The most probable solubility of the pseudoglobulin of serum was found to be 0.07 gm. in 1 liter; of tuberin 0.1 gm. and of casein 0.11 gm. The methods that were employed in their determination are described. 3. Each protein investigated dissolved in water to a constant and characteristic extent when the amount of protein precipitate with which the solution was in heterogeneous equilibrium was varied within wide limits. The solubility of a pure protein is therefore proposed as a fundamental physicochemical constant, which may be used in identifying and in classifying proteins. 4. The concentration of protein dissolved must be the sum of the concentration of the undissociated protein molecule which is in heterogeneous equilibrium with the protein precipitate, and of the concentration of the dissociated protein ions. 5. The dissociated ions of the dissolved protein give a hydrogen ion concentration to water that is also a characteristic of each protein.     

WATER RELATIONS IN THE CELL : I. THE CHLOROPLASTS OF NITELLA AND OF SPIROGYRA

Chloroplasts may contract under natural conditions and give up water to the rest of the cell, thus indicating changes in metabolism or constitution. Such contractions may be produced experimentally. In Nitella the chloroplasts are ellipsoid bodies which, under natural conditions, may contract to spheres with a loss of volume. This may be brought about by lead acetate, ferric chloride, and digitonin: the contraction may occur while the cell is alive. The contraction in lead acetate is reversible (in lead nitrate little or no contraction occurs). In Spirogyra the chloroplast is a long, spirally coiled ribbon which may contract under natural conditions to a short nearly straight rod with a loss of volume. This can be brought about by inorganic salts and in other ways while the cell is still alive.     

THE CHLOROPHYLL-PROTEIN COMPLEX : I. ELECTROPHORETIC PROPERTIES AND ISOELECTRIC POINT

1. Reported effects of different conditions on the stability of the purified chlorophyll-protein complex have been confirmed. 2. The electrophoretic behavior of the chlorophyll-protein complex prepared from two unrelated species of plants (Aspidistra elatior and Phaseolus vulgaris) has been investigated and shown to be dissimilar. In M/50 acetate buffer at 25°C, the isoelectric point of the complex from Phaseolus is at pH 4.70, whereas that from Aspidistra is at pH 3.9 (extrapolated). These values fall within the usual range of protein isoelectric points. 3. Treatment with weak acids causes an irreversible denaturation of the protein complex from both species, with a resultant shift in the mobility-pH curves to more basic values. 4. Differences in electrophoretic behavior between the chlorophyll-protein complex and the cytoplasmic proteins of Phaseolus have been demonstrated. The isoelectric point of the latter is at pH 4.22.     

THE ISOELECTRIC POINTS OF THE PROTEINS IN CERTAIN VEGETABLE JUICES

The state in which a protein substance exists depends upon the nature of its combination with acids or bases and is changed by change in the protein compound. The nature of the compound of a protein that exists at any hydrogen ion concentration can be ascertained if the isoelectric point of the protein is known. Accordingly information regarding the isoelectric points of vegetable proteins is of importance for operations in which it may be desirable to change the state of protein substances, as in the dehydration of vegetables. The Protein in Potato Juice.—The hydrogen ion concentration of the filtered juice of the potato is in the neighborhood of 10^–7 N. Such juice contains the globulin tuberin to the extent of from 1 to 2 per cent. The character of the compound of tuberin that exists in nature was suggested by its anodic migration in an electric field. The addition of acid to potato juice dissociated this compound and liberated tuberin at its isoelectric point. The isoelectric point of tuberin coincided with a slightly lower hydrogen ion concentration than 10^–4 N. At that reaction it existed most nearly uncombined. The flow of current during cataphoresis was greatest in the neighborhood of the isoelectric point. This evidence supplements that of the direction of the migration of tuberin, since it also suggests the existence of the greatest number of uncombined ions near this point. At acidities greater than the isoelectric point tuberin combined with acid. The compound that was formed contained nearly three times as much acid as was needed to dissociate the tuberin compound that existed in nature. At such acidities tuberin migrated to the cathode. Though never completely precipitated tuberin was least soluble in the juice of the potato in the neighborhood of its isoelectric point. Both the compounds of tuberin with acids and with bases were more soluble in the juice than was uncombined tuberin. The nature of the slight precipitate that separated when potato juice was made slightly alkaline was not determined. The Protein in Carrot Juice.—The isoelectric point of the protein in carrot juice coincided with that of tuberin. Remarkably similar also were the properties of carrot juice and the juice of the potato. Existing in nature at nearly the same reaction they combined with acids and bases to nearly the same extent and showed minima in solubility at the same hydrogen ion concentrations. The greatest difference in behavior concerned the alkaline precipitate which, in the carrot, was nearly as great as the acid precipitate. The Protein in Tomato Juice.—The protein of the tomato existed in a precipitated form near its isoelectric point. Accordingly it was not present to any extent in filtered tomato juice. If, however, the considerable acidity at which the tomato exists was neutralized the protein dissolved and was filterable. It then migrated to the anode in an electric field. The addition of sufficient acid to make the hydrogen ion concentration slightly greater than 10^–5 N again precipitated the protein at its isoelectric point. At greater acidities migration was cathodic.     

白鱀豚饲养池水质状况的研究

白鱀豚是我国特有的稀珍水生哺乳动物。仅分布于长江的中、下游干流之中。关于如何饲养白鱀豚没有可借鉴的资料。在没有水净化装置和流水系统的条件下,为了既能节省人力和财力,又能保证豚体的健康,我们对白鱀豚饲养池的理化和微生物因子进行了系统、全面的测定和研究。       

HOMEOSTATIC ADJUSTMENTS AFTER EXERCISE : I. ACID-BASE EQUILIBRIUM OF THE BLOOD

The rate at which displacement and recovery of the acid-base equilibrium of the blood occur in young adult males subjected to short periods of maximal exertion has been determined. Displacement of acid-base equilibrium produced by severe exercise is along the fixed acid path, similar to the path of displacement produced by ingestion of acidifying agents such as ammonium chloride. Maximum displacement of the acid-base equilibrium is not reached until 7 to 10 minutes after the cessation of exercise. By this time over 50 per cent of the displacement in oxygen consumption, respiratory volume, and blood pressure have disappeared. A much greater metabolic acidosis was produced by exercise than could be induced by the oral administration of ammonium chloride. Recovery from the metabolic acidosis produced by exercise was much more rapid (10 times) than was recovery from the acidosis produced by ammonium chloride. After exercise the pH, returned to normal values more rapidly than did the bicarbonate content of the serum.     

THE KINETICS OF OSMOTIC SWELLING IN LIVING CELLS

The rate of swelling of unfertilized sea urchin eggs in hypotonic sea water was investigated. Analysis of curves leads to the following conclusions. 1. The rate of swelling follows the equation, See PDF for Equation where V _eq., V ₀, and V_t stand for volume at equilibrium, at first instant, and at time t, respectively, the other symbols having their usual significance. This equation is found to hold over a wide range of temperatures and osmotic pressures. This relation is the one expected in a diffusion process. 2. The rate of swelling is found to have a high temperature coefficient (Q ₁₀ = 2 to 3, or µ = 13,000 to 19,000). This deviation from the usual effect of temperature on diffusion processes is thought to be associated with changes in cell permeability to water. The possible influence of changes in viscosity is discussed. 3. The lower the osmotic pressure of the solution, the longer it takes for swelling of the cell. Thus at 15° in 80 per cent sea water, the velocity constant has a value of 0.072, in 20 per cent sea water, of 0.006.     

THE DETERMINATION OF ESCHERICHIA COLI I IN SEA WATER

SUMMARY: For the determination of Escherichia coli I in sea water lactose broth frequently gave higher presumptive and confirmed counts than MacConkey's broth. In the presumptive count there were 53 cases where lactose broth gave larger numbers than MacConkey's broth, with 11 equal counts, and only 25 cases with smaller counts ( P =0·00137). After confirmation the corresponding numbers of cases were 39, 10 and 27 ( P =0·134).
In the samples giving most probable numbers (MPN) of less than 100 E. coli /100 ml lactose broth was superior to MacConkey's broth ( P =0·021). At higher MPN values both media were satisfactory, but with highly polluted water MacConkey's broth might give better recoveries due to the suppression of high concentrations of non-coli-aerogenes bacteria.
Samples stored for 24 or 48 hr before testing gave higher presumptive recoveries when examined with lactose broth than with MacConkey's broth, the values for P being 0·028 and 0·0027 respectively.
It appears that lactose broth without inhibitory ingredients could be used with advantage in the examination of sea water.     

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.