THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : II. THE EFFECT OF DIFFUSION GRADIENTS BROUGHT ABOUT BY EGGS IN CAPILLARY TUBES

1. When a Fucus egg develops near one end in a close fitting capillary tube of pyrex glass or silica (quartz), diffusion of substances passing to and from the egg is more impeded on the side of the egg toward the far end of the tube. 2. The egg therefore develops in a gradient of its own diffusion products, and of oxygen tension. 3. More than 600 eggs have been reared, each near one end in a capillary, in sea water at various regulated and measured pH values. 4. When the medium, which is initially homogeneous inside and outside the capillary, is initially at pH 6.5 to 7.6, nearly all of the eggs develop rhizoid protuberances on the sides of the eggs toward the far ends of the capillaries. This is on the sides of the eggs where the concentration of substances diffusing from the eggs is greatest. 5. The polarity and developmental pattern of the egg is thus determined either by a concentration gradient of products diffusing from it, or by a gradient of oxygen tension. The former interpretation is favored. 6. This is regarded as an extension of earlier observations that rhizoid protuberances form on the sides of two neighboring eggs in the direction of the neighbor if the sea water is acidified. 7. It appears hardly possible that a mitogenetic effect could be responsible for the response of an egg to its own diffusion gradients. 8. When the medium is made more basic, the percentage of the eggs which form rhizoid protuberances toward the far end of the tube decreases to about 20 or 25 per cent between pH 8.1 and 8.6. The response of the egg to the gradients which it produces is thus statistically reversed. The determination of the polarity of the eggs by the diffusion gradients does not become as complete in alkalinized as in acidified sea water. 9. When the pH of the sea water is elevated to 9.1 or 9.2, salts precipitate out. The type of development is altered and the control of the diffusion gradients over the polarity of the eggs decreases.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : I. INCREASED HYDROGEN ION CONCENTRATION AND THE INTENSITY OF MUTUAL INDUCTIONS BY NEIGHBORING EGGS OF FUCUS FURCATUS

1. The eggs of Fucus furcatus develop perfectly in sea water acidified to pH 6.0. They are retarded at pH 5.5. At pH 5.0 they do not develop, nor do they cytolize. 2. In normal sea water in the dark at 15°C., eggs develop rhizoids on the sides in the resultant direction of a mass of neighboring eggs. The polarity and the whole developmental pattern of the embryo is thereby induced. This inductive effect does not operate, however, unless the directing mass is an appreciable aggregation of cells (10 or more), or unless there are numerous other eggs in the dish. A group of five eggs alone in a dish do not carry out mutual inductions. Two eggs alone in a dish do not develop rhizoids toward each other. 3. When the sea water is acidified to pH 6.0 all sizes of aggregations carry out mutual inductions. Two eggs alone in a dish now develop rhizoids on the sides toward each other, provided they are not more than about 4 egg diameters apart. 4. Increased hydrogen ion concentration thus augments or intensifies the mutual inductive effect. 5. This may explain why only larger masses of eggs show inductions in normal sea water, since presumably the larger masses considerably increase the hydrogen ion concentration locally. 6. The nature of the inductive action is discussed. 7. In acidified sea water at pH 6.0, compared with normal sea water at pH 7.8–8.0, the rhizoids originate and extend with a strongly increased downward component. The substrate then forces further extension or growth of the rhizoid to be in the plane of the substrate.     

THE PHYSIOLOGY OF THE RESPIRATION OF FISHES IN RELATION TO THE HYDROGEN ION CONCENTRATION OF THE MEDIUM

1. The ability of marine fishes to absorb oxygen at low tension from the sea water is more or less dependent upon the hydrogen ion concentration of the water. 2. The ability of fishes to withstand wide variations in the range of hydrogen ion concentration of the sea water can be correlated with their habitats. The fishes that are most resistant to a wide variation in the hydrogen ion concentration are most cosmopolitan in their habitat. Those that are least resistant to a variation in the hydrogen ion concentration are the most restricted in their range of habitat. 3. There is a close correlation between the optimum condition of the sea water for the absorption of oxygen at low tension by the herring (Clupea pallasii), the condition of the sea water to which they react positive and that in which they are found most abundantly. 4. It is suggested that the variation in the ability to absorb oxygen at low tension at a given pH of individuals of a species is dependent upon the alkaline reserve of the blood of the individual fish.     

THE EFFECT OF HYDROGEN ION CONCENTRATION ON SWELLING OF CELLS

1. The effect of HCl, NaOH, CO₂, and NH₃ on the volume of unfertilized Arbacia eggs was tested over a wide range of pH values. 2. No swelling occurred, except in HCl solutions, and there not until after injury or death had occurred. 3. Whereas the volume of erythrocytes and of proteins such as gelatin is known to be dependent on the pH of the solution, such a relation does not exist in the case of living and uninjured cells, at least of the type tested.     

CHEMICAL CHARACTER AND PHYSIOLOGICAL ACTION OF THE POTASSIUM ION

1. It is shown that the NH₄ ion acts in cases of antagonism on the egg of Fundulus more like the K ion than the Na ion; this corresponds to the fact that in its general chemical behavior the NH₄ ion resembles the K ion more closely than the Na ion. 2. It is shown that the tolerance of sea urchin eggs towards the Li ion can be increased 500 per cent or more if at the same time a certain amount of Na ion is replaced by K, Rb, or Cs ions. Since in the periodic table Na occupies a position between K and Li it is inferred that the Li and K ions deviate in their physiological action in the opposite direction from the Na ion. 3. These data indicate that the behavior of the K ion in antagonistic salt action (which forms the basis of the physiologically balanced action of ions) is due to its purely chemical character, i.e. its position in the periodic table or rather to its atomic number, and not to those explosions in its nucleus which give rise to a trace of radioactivity.     

REGULATION OF THE HYDROGEN ION CONCENTRATION AND ITS RELATION TO METABOLISM AND RESPIRATION IN THE STARFISH

The normal reaction of the cœlomic fluid in Patiria miniata and Asterias ochraceus is pH 7.6, and of the cæca, 6.7, compared with sea water at 8.3, all without salt error correction. A medium at pH 6.7–7.0 is optimum for the cæca for ciliary survival and digestion of protein, and is maintained by carbon dioxide production. The optimum pH found for carbon dioxide production is a true one for the effect of hydrogen ion concentration on the tissue. It does not represent an elimination gradient for carbon dioxide. Because the normal excised cæca maintain a definite hydrogen ion concentration and change their internal environment toward that as an optimum during life, there exists a regulatory process which is an important vital function.     

THE INFLUENCE OF HYDROGEN ION CONCENTRATION ON THE INACTIVATION OF PEPSIN SOLUTIONS

1. Pepsin in solution at 38°C. is most stable at a hydrogen ion concentration of about 10^–5 (pH 5.0). 2. Increasing the hydrogen ion concentration above pH 5.0 causes a slow increase in the rate of destruction of pepsin. 3. Decreasing the hydrogen ion concentration below pH 5.0 causes a very rapid increase in the rate of destruction of the enzyme. 4. Neither the purity of the enzyme solution nor the anion of the acid used has any marked effect on the rate of destruction or on the zone of hydrogen ion concentration in which the enzyme is most stable. 5. The existence of an optimum range of hydrogen ion concentration for the digestion of proteins by pepsin cannot be explained by the destruction of the enzyme by either too weak or too strong acid.     

THE SIGNIFICANCE OF THE HYDROGEN ION CONCENTRATION FOR THE DIGESTION OF PROTEINS BY PEPSIN

The experiments described above show that the rate of digestion and the conductivity of protein solutions are very closely parallel. If the isoelectric point of a protein is at a lower hydrogen ion concentration than that of another, the conductivity and also the rate of digestion of the first protein extends further to the alkaline side. The optimum hydrogen ion concentration for the rate of digestion and the degree of ionization (conductivity) of gelatin solutions is the same, and the curves for the ionization and rate of digestion as plotted against the pH are nearly parallel throughout. The addition of a salt with the same anion as the acid to a solution of protein already containing the optimum amount of the acid has the same depressing effect on the digestion as has the addition of the equivalent amount of acid. These facts are in quantitative agreement with the hypothesis that the determining factor in the digestion of proteins by pepsin is the amount of ionized protein present in the solution. It was shown in a previous paper that this would also account for the peculiar relation between the rate of digestion and the concentration of protein. The amount of ionized protein in the solution depends on the amount of salt formed between the protein (a weak base) and the acid. This quantity, in turn, according to the hydrolysis theory of the salts of weak bases and strong acids, is a function of the hydrogen ion concentration, up to the point at which all the protein is combined with the acid as a salt. This point is the optimum hydrogen ion concentration for digestion, since the solution now contains the maximum concentration of protein ions. The hydrogen ion concentration in this range therefore is merely a convenient indicator of the amount of ionized protein present in the solution and takes no active part in the hydrolysis. After sufficient acid has been added to combine with all the protein, i.e. at pH of about 2.0, the further addition of acid serves to depress the ionization of the protein salt by increasing the concentration of the common anion. The hydrogen ion concentration is, therefore, no longer an indicator of the amount of ionized protein present, since this quantity is now determined by the anion concentration. Hence on the acid side of the optimum the addition of the same concentration of anion should have the same influence on the rate of digestion irrespective of whether it is combined with hydrogen or some other ion (provided, of course, that there is no other secondary effect of the other ion). The proposed mechanism is very similar to that suggested by Stieglitz and his coworkers for the hydrolysis of the imido esters. Pekelharing and Ringer have shown that pure pepsin in acid solution is always negatively charged; i.e., it is an anion. The experiments described above show further that it behaves just as would be expected of any anion in the presence of a salt containing the protein ion as the cation and as has been shown by Loeb to be the case with inorganic anions. Nothing has been said in regard to the quantitative agreement between the increasing amounts of ionized protein found in the solution (as shown by the conductivity values) and the amount predicted by the hydrolysis theory of the formation of salts of weak bases and strong acids. There is little doubt that the values are in qualitative agreement with such a theory. In order to make a quantitative comparison, however, it would be necessary to know the ionization constant of the protein and of the protein salt and also the number of hydroxyl (or amino) groups in the protein molecule as well as the molecular weight of the protein. Since these values are not known with any degree of certainty there appears to be no value at present in attempting to apply the hydrolysis equations to the data obtained. It it clear that the hypothesis as outlined above for the hydrolysis of proteins by pepsin cannot be extended directly to enzymes in general, since in many cases the substrate is not known to exist in an ionized condition at all. It is possible, however, that ionization is really present or that the equilibrium instead of being ionic is between two tautomeric forms of the substrate, only one of which is attacked by the enzyme. Furthermore, it is clear that even in the case of proteins there are difficulties in the way since the pepsin obtained from young animals, or a similar enzyme preparation from yeast or other microorganisms, is said to have a different optimum hydrogen ion concentration than that found for the pepsin used in these experiments. The activity of these enzyme preparations therefore would not be found to depend on the ionization of the protein. It is possible of course that the enzyme preparations mentioned may contain several proteolytic enzymes and that the action observed is a combination of the action of several enzymes. Dernby has shown that this is a very probable explanation of the action of the autolytic enzymes. The optimum hydrogen ion concentration for the activity of the pepsin used in these experiments agrees very closely with that found by Ringer for pepsin prepared by him directly from gastric juice and very carefully purified. Ringer''s pepsin probably represents as pure an enzyme preparation as it is possible to prepare. There is every reason to suppose therefore that the enzyme used in this work was not a mixture of several enzymes.     

THE EFFECT OF HYDROGEN ION CONCENTRATION ON THE PRODUCTION OF CARBON DIOXIDE BY BACILLUS BUTYRICUS AND BACILLUS SUBTILIS

1. The maximum rate of CO₂ production of Bacillus butyricus was found to be at a pH value of 7; of Bacillus subtilis at pH 6.8. If the pH value be raised or lowered there is a progressive decrease in the rate of production of CO₂. 2. Spontaneous recovery follows the addition of alkali to either organism, while addition of acid is followed by recovery only upon addition of an equivalent amount of alkali, and is not complete except when the amount of acid is very small.     

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 R?LE OF THE ACTIVITY COEFFICIENT OF THE HYDROGEN ION IN THE HYDROLYSIS OF GELATIN

1. The hydrolysis of gelatin at a constant hydrogen ion concentration follows the course of a monomolecular reaction for about one-third of the reaction. 2. If the hydrogen ion concentration is not kept constant the amount of hydrolysis in certain ranges of acidity is proportional to the square root of the time (Schütz''s rule). 3. The velocity of hydrolysis in strongly acid solution (pH less than 2.0) is directly proportional to the hydrogen ion concentration as determined by the hydrogen electrode i.e., the "activity;" it is not proportional to the hydrogen ion concentration as determined by the conductivity ratio. 4. The addition of neutral salts increases the velocity of hydrolysis and the hydrogen ion concentration (as determined by the hydrogen electrode) to approximately the same extent. 5. The velocity in strongly alkaline solutions (pH greater than 10) is directly proportional to the hydroxyl ion concentration. 6. Between pH 2.0 and pH 10.0 the rate of hydrolysis is approximately constant and very much greater than would be calculated from the hydrogen and hydroxyl ion concentration. This may be roughly accounted for by the assumption that the uncombined gelatin hydrolyzes much more rapidly than the gelatin salt.     

THE EFFECTS OF HYDROGEN ION CONCENTRATION UPON THE METAMORPHIC PATTERN OF THYROXIN- AND IODINE-TREATED TADPOLES

1. It has been shown quantitatively that the degree of response of the hind limbs of tadpoles to the action of thyroxin is dependent upon the lengths of the limbs at the beginning of treatment. 2. Both the potency of the inducing substance and the rate of penetration of the substance into the animal might be involved in the effects of hydrogen ion concentration on induced development. 3. Changes in hydrogen ion concentration affect the inducing power of thyroxin and iodine differently. With thyroxin, it is the rate of penetration of the molecule which determines the amount of growth, but with iodine it is the chemical form in which the substance has entered the animal which is of prime importance. 4. The hydrogen ion concentration of thyroxin solutions does not affect their potency when they are injected into tadpoles. 5. Change in hydrogen ion concentration of the environment does not affect the potency of thyroxin injected into tadpoles. 6. When thyroxin is administered in the environmental solution its effects, as measured by increase in hind limb length are greater at higher than at lower hydrogen ion concentrations in the range tested. 7. Since the potency of thyroxin is unaffected by change in hydrogen ion concentration when the thyroxin solution is injected, the above fact (point 6) seems explicable only on the basis of differences in the rate of penetration of thyroxin into the animals at the different hydrogen ion concentrations. 8. These differences in penetration of the thyroxin at different hydrogen ion concentrations may be the result of a differential effect of hydrogen ion concentration upon the rate of metabolism of the animal. The metabolic rate is significantly greater when the tadpoles are kept in solutions of higher hydrogen ion concentration than when they are kept in solutions of low hydrogen ion concentration. It is postulated that the rate of metabolism, since it controls the rate of intake of the environmental fluid and therefore of dissolved thyroxin, also controls the amount of thyroxin-induced development. 9. Change in hydrogen ion concentration of iodine solutions affects their potency when injected into tadpoles. A peak of effectiveness is reached at about the neutral point, with a lowered efficiency as the hydrogen ion concentration is either increased or decreased from this point. 10. Change in hydrogen ion concentration of the environment affects the potency of iodine injected into tadpoles. The effect is similar to that noted in point 9. 11. The hydrogen ion concentration of the environment seems to affect the chemical nature of the iodine in solution in the environment. If this is so, it is possible that the differences in the metamorphic effects of iodine at different hydrogen ion concentrations are dependent upon the chemical form of iodine present. 12. The effect of hydrogen ion concentration on normal development is similar to that on thyroxin-induced development; an effect on the rate of metabolism of the animal causes increased growth in more acid solutions.     

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).     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

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.     

THE EFFECT OF THE H ION CONCENTRATION ON THE AVAILABILITY OF IRON FOR CHLORELLA SP

The data obtained in these experiments indicate clearly that unless the necessary precautions are taken to keep the iron of the culture medium in solution the results obtained by varying the H ion concentration will not represent the true effect of this factor on growth. The availability of iron in nutrient solutions has been the subject of numerous recent investigations and it is now known that iron is precipitated at the lower hydrogen ion concentrations, that the iron of certain iron salts is less likely to be precipitated than that of others, and that certain salts of organic acids tend to keep the iron in solution. In general, ferric citrate seems to be the most favorable source of iron. In addition to chemical precipitation, however, it is also possible for the iron to be removed by adsorption on an amorphous precipitate such as calcium phosphate. As this precipitate is frequently formed when nutrient solutions are made alkaline, this may account for the discordant results reported in the literature as to the availability of certain forms of iron. By omitting calcium from the culture solution iron can be maintained in a form available for growth in alkaline solutions by the addition of sodium citrate. In such solutions the maximum growth of Chlorella occurred at pH 7.5. The alkaline limit for growth has not been established as yet. In investigating the availability of iron at varying concentrations of the hydrogen ion, changes in the pH value of the solution during the course of an experiment should also be taken into account. This is especially important in unbuffered solutions. The differential absorption of the ions of ammonium salts may cause a marked increase in the hydrogen ion concentration, which in turn will cause an increase in the solubility of iron. In strongly buffered solutions as used in these experiments this effect is slight.     

INFLUENCE OF TEMPERATURE AND HYDROGEN ION CONCENTRATION UPON THE SPORE CYCLE OF BACILLUS SUBTILIS

1. At 5°C. no germination took place. 2. At 25°C. and at 37°C. germination occurs if the hydrogen ion concentration of the broth is kept between pH 5 and pH 10, but not at higher or lower pH values. 3. The completion of the spore cycle likewise requires a hydrogen ion concentration between pH 5 and pH 10. 4. The spores can germinate when the pH value is 10, although after germination the vegetative cells multiply only to a very slight extent and soon pass into spores. 5. The slight growth and multiplication of vegetative cells in broth of pH 10 suggest that the formation of endospores in this medium must be caused largely by the unfavorable reaction of the medium rather than by the accumulation of metabolic products. 6. Automatic adjustment of the medium seems to play a rôle in the completion of the spore cycle. 7. The results are not only of theoretical importance but they have a practical application to the preservation of food by canning and by other methods.     

THE EQUILIBRIUM BETWEEN HEMOLYTIC SENSITIZER AND RED BLOOD CELLS IN RELATION TO THE HYDROGEN ION CONCENTRATION

1. In a salt-free medium the proportion of the total amount of hemolytic sensitizer present, combined with the homologous cells, reaches a maximum of almost 100 per cent at pH 5.3. On the alkaline side of this point the proportion combined diminishes with the alkalinity and reaches a minimum of approximately 5 per cent at pH 10. On the acid side of pH 5.3 the proportion combined diminishes with the acidity but somewhat less rapidly than for a corresponding increase in alkalinity. 2. The presence of NaCl greatly increases the proportion of sensitizer combined with cells at all reactions except those in the neighborhood of pH 5.3. At this point the combination of sensitizer with cells is independent of the presence of electrolyte. 3. The curves representing the proportion of sensitizer combined or free run almost exactly parallel, both when the sensitizer combines de novo and when it dissociates from combination; therefore, in constant volume, at a given hydrogen ion concentration, and at a given temperature, an equilibrium exists between the amount of sensitizer free and that combined with cells. 4. The combination of sensitizer and cells is related fundamentally to the isoelectric point of the sensitizer. 5. The dissociated ions of the sensitizer, formed either by its acid or its basic dissociation, do not unite with cells. Combination takes place only between the cells and the undissociated molecules of the sensitizer.