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 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.     

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 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.     

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 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 CAMBIUM AND ITS DERIVATIVE TISSUES : VI. THE EFFECTS OF HYDROGEN ION CONCENTRATION IN VITAL STAINING

It should be emphasized, in conclusion, that the writers'' investigation is a reconnaissance, and was initiated primarily in searching for more adequate techniques for the study of cytological problems. Crude as many of the data undoubtedly are, they are of some significance in outlining future trends of more intensive investigation. The occurrence of two distinct types of vacuoles within the same cell provides a valuable check upon generalizations concerning the penetration of certain dyes. The A-type vacuole affords a means of determining that a number of dyes do penetrate living plant cells readily and rapidly from acid buffers. The recognition of two distinct categories of vacuoles—which are widely distributed throughout the higher plants—and a study of their staining reactions in Group I, Group II, and Group III dyes, indicate that certain discrepancies in the literature are due to the fact that different investigators are concerned with different vacuoles and with different dyes. For an accurate visualization of the physico-chemical mechanisms of the penetration and accumulation of dyes in living cells a much wider range of reliable data is essential, both as regards the purely biological variables and the physico-chemical variables in techniques employed in their investigation. Until such data are available, generalizations from limited induction should be reduced to a minimum.     

THE COMBINATION OF ENZYME AND SUBSTRATE : I. A METHOD FOR THE QUANTITATIVE DETERMINATION OF PEPSIN. II. THE EFFECT OF THE HYDROGEN ION CONCENTRATION.

1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein.     

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.     

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.     

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.     

HYDROGEN ION CONCENTRATIONS IN THE BLOOD OF INSECTS

The range of pH values for the blood of grasshoppers and of houseflies is 7.2 to 7.6. The range of values for roaches is 7.5 to 8.0. The range for Malacosoma americanum is 6.4 to 7.4; and the range for Bombyx mori is 6.4 to 7.2. From the work of other investigators and from the writer''s results, it is apparent that the pH of insect blood, in general, may vary between 6.4 and 8.0. In the forms observed no correlation exists between blood pH and age, nor between pH and metamorphosis.     

The Geoelectric Effect in Plant Shoots: III. DEPENDENCE UPON AUXIN CONCENTRATION GRADIENTS AND AEROBIC METABOLISM

The development of the geoelectric effect has been followedin Zea coleoptiles with a flowing-solution electrode system,and its dependence upon auxin concentration gradients and aerobicmetabolism assessed. A symmetrical source of IAA can effectively replace the coleoptiletip in allowing the geo-electric potential to occur. The diffusatefrom coleoptile tips, when applied asymmetrically to the apexof a vertical decapitated coleoptile, generates a potentialdifference across the coleoptile indistinguishable from thatinduced by the asymmetrical application of IAA. Asymmetricalapplication of IAA to vertical Avena and Zea coleoptiles andHelianthus hypocotyls induces closely similar responses. Neither the geoelectric effect nor a geotropic response developswhen intact Zea coleoptiles are placed horizontally after beingdeprived of oxygen, but they both occur when an aerobic atmosphereis restored. The lateral potential difference induced by theasymmetrical application of IAA to the apex of a vertical coleoptiledoes not occur under anoxic conditions. With a static-drop electrode system and a decapitated Zea coleoptile,a potential difference develops immediately after reorientationof the coleoptile into the horizontal position, and attainsa maximum value after about 10 min. This potential differencecan be further increased by the asymmetrical application ofIAA to the lower half of the apical cut surface of the coleoptile. Our data support the view that both the geoelectric potentialand the geotropic response are due to the IAA concentrationgradient which arises from the lateral transport of this substancefrom the upper to the lower half of the horizontal shoot. Theyalso bear out our previous conclusions that the ‘geoelectricpotential’ observed with static-drop electrodes and anintact shoot, is the resultant of two processes. The first isa physical phenomenon arising in the electrodes, or betweenthe electrodes and the plant tissue, and the second arises inthe living tissues of the shoot as the result of gravity-inducedchanges in auxin distribution.