ELECTROPHORESIS OF BACTERIA AS INFLUENCED BY HYDROGEN ION CONCENTRATION AND THE PRESENCE OF SODIUM AND CALCIUM SALTS

1. We have confirmed the results of earlier workers particularly of Northrop and De Kruif in regard to the following points: (a) the general tendency of the bacterial cell when suspended in distilled water near the zone of neutrality to move toward the anode of an electrical field; (b) the fact that the migration of bacterial cells in the electrical field is a function of the reaction of the menstruum. The curve obtained by plotting velocity of migration against pH passes through an isoelectric point at about pH 3.0, at greater acidity the direction of migration becomes reversed (toward the cathode) and in still more acid solution (pH = 1.0) again disappears; while at reactions less acid than pH 3.0 the velocity is toward the anode and increases with increasing alkalinity; (c) the fact that neutral salts depress the velocity of migration, calcium salts being much more effective than sodium salts of the same concentration. 2. We further find: (a) that on the extreme alkaline side of the curve of velocity of migration plotted against pH a maximum value is reached at about pH 10 with a fall at about pH 12.0 which in many experiments reaches an isopotential point; (b) that the depressing effect of salts is accompanied by a general shifting of the curve of migration velocity so that a maximum velocity (of course absolutely less than that manifest in the absence of salts) appears at about pH 7.0 and an abolition of velocity at pH 9.0 to 10.0; (c) that an apparent "antagonistic" effect is indicated between CaCl₂ and NaCl, the presence of a certain concentration of the latter salt diminishing to a slight but definite degree the depressing effect produced by the former; (d) that heat-killed bacterial cells exhibit essentially the same curve of migration velocity as that of the living cells; (e) that bacterial spores exhibit the same general curve of migration velocity as vegetative cells, although the actual velocity is apparently slightly less. 3. All of the observed phenomena appear to be in accord with the assumption that marked differences in dielectric constants did not appear under the conditions studied and if this assumption be granted the results are in accord with the fundamental postulates of the Donnan equilibrium as applied to the explanation of the origin of potential difference between a bacterial cell and its enveloping menstruum. It is possible but not at all certain that the phenomenon of antagonism may require the introduction of additional assumptions for its explanation. Professor Donnan and other investigators have clearly understood the importance of applying the concept of membrane equilibria in the elucidation of physiological phenomena. Our findings add to the numerous vindications favoring this view and emphasize the importance of further study of membrane equilibria in bacterial suspensions. We have pointed out that certain potential differences between bacteria and their menstrua are apparently associated with some of the phenomena of viability. Viability and potential differences may, however, under certain conditions vary quite independently as evidenced by the fact that normal rates of migration are demonstrable after the cells have been killed by heat. Thus, considerable caution must be exercised in relating the existence of these charges to the metabolism of the cell.     

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.     

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.     

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 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.     

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.     

CONDUCTIVITY AS A MEASURE OF THE PERMEABILITY OF SUSPENDED CELLS

The problem of determining by means of measurements of electrolytic conductance the permeability of living cells in suspension is considered in some detail and it is pointed out that several factors, usually neglected, have an important influence on the interpretation of such studies. These are: 1. The relative volume and the shape of cells, which are responsive to changes in osmotic pressure and constitution of the surrounding solution. The sources of error in various methods of determining the true volume of red blood cells in a suspension are explained. The hematocrit method appears to be the most reliable method in this case. 2. The proportion of living cells, which is especially to be regarded in the case of suspensions of bacteria. It is shown that this may be very high when appropriate cultural methods are used. The conductance of the dead cells must also be taken into account. 3. The progressive nature of the changes occurring during the course of an experiment. Approximate accuracy may be obtained by proper interpolation. 4. The conductivity of the protoplasm itself, which varies in response to variations is that of the surrounding fluid. It is emphasized that cells, and in particular red blood cells, are not to be regarded as stable non-conducting particles, but rather as labile and as permeable to electrolytes. It is shown that the available data support this interpretation.     

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.