EFFECT OF THE HYDROGEN ION CONCENTRATION ON THE PHAGOCYTOSIS AND ADHESIVENESS OF LEUCOCYTES

1. Immediately after coming into contact with glass, leucocytes are most adhesive at pH 8.0 or > 8.0. 2. Agglutination of leucocytes increases with increasing H ion concentration from pH 8.0 to 6.0. 3. In phagocytosis experiments where leucocytes creep about on the slide picking up articles the optimum pH is 7.0. Here ameboid movement is probably the limiting factor. 4. The optimum for phagocytosis of quartz from suspension is on the acid side of neutrality at or near pH 6.7. 5. Phagocytosis of quartz increases with the acidity, while adhesiveness of leucocytes to glass increases with the alkalinity.     

THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE INDUCTION OF POLARITY IN FUCUS EGGS : III. GRADIENTS OF HYDROGEN ION CONCENTRATION

1. Gradients of hydrogen ion concentration across Fucus eggs growing in sea water determine the developmental polarity of the embryo. 2. Gradients may determine polarity even if removed before the morphological response begins. 3. The rhizoid forms on the acid side of the egg unless this is too acid, in which case it develops on the basic side of the egg. 4. Since gradients of hydrogen ion concentration in sea water produce gradients of CO₂ tension, as a result of chemical action on the carbonate buffer system, it is not proven whether the physiological effects are due to the hydrogen ions, or to the CO₂ which they produce in the medium. 5. The developmental response of the eggs to gradients of hydrogen ion (or CO₂) concentration provides an adequate but not an exclusive explanation of the group effect in Fucus. 6. Hydrogen ions may exert their effect by activating growth substance. Hydrogen ions or CO₂ probably also affect the underlying rhizoid forming processes in other ways as well.     

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 PERMEABILITY OF LIVING CELLS TO DYES AS AFFECTED BY HYDROGEN ION CONCENTRATION

1. An accurate quantitative method of measuring the penetration of dye into the living cell is described. 2. Cresyl blue is unable to penetrate rapidly unless the pH outside the cell is decidedly greater than that inside. The rate of penetration increases with increasing pH. 3. Around pH 9 penetration of the dye is rapid while the reverse is true of exosmosis. At low pH values (5.9) exosmosis is rapid and penetration is very slow.     

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.     

THE SELECTIVE ABSORPTION OF POTASSIUM BY ANIMAL CELLS : III. THE EFFECT OF HYDROGEN ION CONCENTRATION UPON THE RETENTION OF POTASSIUM.

By perfusing frogs for varying periods with potassium-free Ringer solutions having a pH ranging from 6.0 to 8.0, it has been determined that such solutions have little or no effect upon the retention of potassium by muscle cells.     

COMPARATIVE STUDIES ON RESPIRATION : XI. THE EFFECT OF HYDROGEN ION CONCENTRATION ON THE RESPIRATION OF PENICILLIUM CHRYSOGENUM.

1. Variations in pH value between 4 and 8 produce practically no effect on the normal rate of respiration (the rate at neutrality is called normal). 2. Increasing the pH value to 8.80 causes respiration to fall to 60 per cent of the normal, after which it remains stationary for the duration of the experiment. 3. Decreasing the pH value to 2.65 causes a gradual rise and a gradual return to normal; at pH 1.10 to 1.95 the preliminary rise amounts to 20 per cent and is followed by a fall to below the normal. 4. The decrease in respiration brought about by solutions of a pH value of 1.95 or less are irreversible, while a similar decrease which occurs at pH 8.80 is reversible, the rate coming back to practically normal after the material is replaced in a neutral solution. 5. Determinations by means of Winkler''s method showed an increase in the consumption of oxygen in acid solutions and a decrease in alkaline solutions.     

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 PENETRATION OF DYES AS INFLUENCED BY HYDROGEN ION CONCENTRATION

When cells of Nitella are placed in buffer solutions at pH 9, there is a very slow and gradual increase in the pH of the sap from pH 5.6 to 6.4 (when death of the cells takes place). If the living cells are placed in 0.002 per cent dye solutions of brilliant cresyl blue at different pH values (from pH 6.6 to pH 9), it is found that the rate of penetration of the dye, and the final equilibrium attained, increases with increase in pH value, which can be attributed to an increase in the active protein (or other amphoteric electrolyte) in the cell which can combine with the dye.     

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.     

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 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.     

CHANGES IN THE APPARENT CYTOPLASMIC HYDROGEN ION CONCENTRATION OF AMEBA DUBIA ON INJECTION OF EGG ALBUMIN

1. Egg albumin when injected into an ameba or discharged into the solution about it raises the apparent pH of the cytoplasm of the ameba. 2. With time the cytoplasm returns to the original pH 6.9 if the nucleus is present. Amebae that have received repeated injections of albumin in some cases extrude their nuclei. In these cells the cytoplasm remains at the more alkaline pH induced by the albumin for at least 12 hours. 3. When a 2 per cent solution of albumin is introduced into a suspension of amebae there is a temporary marked rise in the rate at which CO₂ is given off with no corresponding rise in O₂ uptake. 4. The results observed can be explained if the albumin discharged onto the surface of the ameba rapidly enters the cell and there becomes distributed in a phase of the cytoplasm other than the one which contains the phenol red.     

THE THERMOLABILITY OF COMPLEMENT,IN RELATION TO THE HYDROGEN ION CONCENTRATION

1. The destruction which complement undergoes on being heated in dilution in distilled water is least at a reaction between pH 6.1 and 6.4. This depends upon the relative preservation of the midpiece function at this point. This reaction represents probably the isoelectric point of a compound of the euglobulin with some substance present also in serum. 2. During the process of thermoinactivation it is chiefly or entirely the ions of this euglobulin compound which react, and these combine or interact with substances contained in the pseudoglobulin and albumin fraction. 3. The behavior of the euglobulin is different in the anionic and in the cationic condition, since on the acid side of pH 6.1 to 6.4 the destruction by heat increases as rapidly with the acidity in the presence as in the absence of NaCl. On the alkaline side of this point the presence of NaCl protects complement from destruction because of the depression in the ionization of the euglobulin.