CHEMICAL ANTAGONISM OF IONS : IV. EFFECT OF SALT MIXTURES ON GLYCINE ACTIVITY.

The pH of a 0.01 molar solution of glycine, half neutralized with NaOH, is 9.685. Addition of only one of the salts NaCl, KCl, MgCl₂, or CaCl₂ will lower the pH of the solution (at least up to 1 µ). If a given amount of KCl is added to a glycine solution, the subsequent addition of increasing amounts of NaCl will first raise the pH (up to 0.007 M NaCl). Further addition of NaCl (up to 0.035 M NaCl) will lower the pH, and further additions slightly raise the pH. The same type of curve is obtained by adding NaCl to glycine solution containing MgCl₂ or CaCl₂ except that the first and second breaks occur at 0.015 M and 0.085 M NaCl, respectively. Addition of CaCl₂ to a glycine solution containing MgCl₂ gives the same phenomena with breaks at 0.005 M and 0.025 M CaCl; or at ionic strengths of 0.015 µCaCl₂ and 0.075 µCaCl₂. This indicates that the effect is a function of the ionic strength of the added salt. These effects are sharp and unmistakable. They are almost identical with the effects produced by the same salt mixtures on the pH of gelatin solutions. They are very suggestive of physiological antagonisms, and at the same time cannot be attributed to colloidal phenomena.     

Specific adsorption of phosphate ions on proteins evidenced by capillary electrophoresis and reversed-phase high-performance liquid chromatography

Specific adsorption of phosphate ions at pH=7.0 was studied on different proteins, either counter-ions of phosphate (lysozyme, lactoferrin) or co-ion of phosphate (α-lactalbumin). The theoretical electrophoretic mobility of globular proteins lysozyme and α-lactalbumin (apo and holo (+1 calcium per molecule) forms) was compared with those measured by capillary electrophoresis in phosphate at pH 7.0, versus the ionic strength (I) in the range 0–0.775 mol L⁻¹. The specific adsorption of phosphate ions was evidenced by difference. From the experimental charge number (Z^eff) of protein in phosphate medium, a phosphate content per protein molecule was determined at pH=7.0.

• •For lactoferrin (pI=8–9), the electrophoretic mobility (μ) was constant and negative, highlighting a charge reversal due to phosphate adsorption.
• •For α-lactalbumin (holo form) experimental μ was roughly constant and more negative than predicted. Z^eff increased continuously from −4 to −11 in the ionic strength range from 0.005 to 0.775 mol l⁻¹, respectively. Accordingly, one to six phosphates were bound per molecule, respectively.
• •For lysozyme, experimental electrophoretic mobility was positive but lower than predicted. Z^eff was only discrete values +5 for I in the range 0.001–0.020 mol l⁻¹ and about +3 in the range 0.050–0.500 mol l⁻¹, whereas the theoretical Z value was +7 at pH=7.0. Lysozyme bounds one phosphate at low ionic strength and about two — three at higher ionic strength.

Reversed-phase HPLC confirms that adsorption of phosphate is different for the three proteins.     

Reversible association processes of globular proteins. I. Insulin

1.

1. The dissociation of insulin is favored by (a) an increase in charge, (b) a decrease in ionic strength, and (c) an increase in temperature.     

CHEMICAL ANTAGONISM OF IONS : III. EFFECT OF SALT MIXTURES ON GELATIN ACTIVITY.

1.25 per cent gelatin solutions containing enough NaOH to bring them to pH 7.367 (or KOH to pH 7.203) were made up with various concentrations of NaCl, KCl and MgCl₂, alone and in mixtures, up to molar ionic strength. The effects of these salts on the pH were observed. MgCl₂ and NaCl alone lower the pH of the Na gelatinate or the K gelatinate, in all amounts of these salts. KCl first lowers the pH (up to 0.01 M K⁺), then raises the pH. Mixtures of NaCl and KCl (up to 0.09 M of the salt whose concentration is varied) raise the pH; then (up to 0.125 M Na⁺ or K⁺) lower the pH; and finally (above 0.125 M) behave like KCl alone. Mixtures of MgCl₂ and NaCl raise the pH up to 0.10 M Na⁺, and lower it up to 0.15 M Na⁺ regardless of the amount of MgCl ₂. Higher concentrations of NaCl have little effect, but the pH in this range of NaCl concentration is lowered with increase of MgCl₂. Mixtures of MgCl₂ and KCl behave as above described (for MgCl₂ and NaCl) and the addition of NaCl plus KCl to gelatin containing MgCl₂ produces essentially the same effect as the addition of either alone, except that the first two breaks in this curve come at 0.07 M and 0.08 M [Na⁺ + K⁺] and there is a third break at 0.12 M. In this pH range the free groups of the dicarboxylic acids and of lysine are essentially all ionized and the prearginine and histidine groups are essentially all non-ionized. The arginine group is about 84 per cent ionized. Hence we are studying a solution with two ionic species in equilibrium, one with the arginine group ionized, and one with it non-ionized. It is shown that the effect of each salt alone depends upon the effect of the cation on the activity of these two species due to combination. The anomalous effects of cation mixtures may be qualitatively accounted for if one or both of these species fail to combine with the cations in a mixture in proportion to the relative combination in solutions of each cation alone. Special precautions were taken to ensure accuracy in the pH measurements. The mother solutions gave identical readings to 0.001 pH and the readings with salts were discarded when not reproducible to 0.003 pH. All doubtful data were discarded.     

Affinity of arginase for ornithine carbamoyltransferase in Saccharomyces cerevisiae.

The association between the two trimeric enzymes ornithine carbamoyltransferase and arginase, which is under the control of arginine and ornithine, is endothermic (ΔH° = + 14.6 kcal mol^?1). The process is clearly entropy-driven (ΔS° = + 94.7 cal mol^?1 deg.^?1) allowing a dissociation constant of 0.1 nm for the complex at optimal pH 8, 30 °C and an ionic strength of 0.025. The stability of the complex is moderately sensitive to pH and ionic strength. The dissociation constant of the complex was measured in a medium at cellular pH and salt concentration and found to be close to the constant expected for operative inhibition of ornithine Carbamoyltransferase in the yeast cell. The importance of the presence of arginine as an effector for the formation of the complex under the above conditions is clearly demonstrated.     

ISOELECTRIC POINTS FOR THE MYCELIUM OF FUNGI

1. Mycelium of Rhizopus nigricans when stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.0. 2. When grown on potato dextrose agar the reaction of which was varied with phosphoric acid the extent of colony growth of Rhizopus nigricans plotted against the initial Sörensen value of the agar produced a double maximum curve with the minimum between the two maxima at initial pH 5.2. 3. When grown in potato dextrose broth the reaction of which was varied with phosphoric acid the dry matter produced by Rhizopus nigricans plotted against the Sörensen value of the broth produced a double maximum curve with the minimum between the two maxima at initial pH 5.2 or average pH 4.9. 4. Mycelium of Rhizopus nigricans placed in buffer mixtures of 0.01 M phosphoric acid and sodium hydroxide of pH 4.1 to 6.3, changed the reaction in most cases toward greater alkalinity. 5. Mycelium of Fusarium lycopersici stained with certain acid and basic dyes and washed with buffer mixtures of 0.1 M phosphoric acid and sodium hydroxide responded much like an amphoteric colloid with an isoelectric point near pH 5.5.     

BIOELECTRIC POTENTIALS IN VALONIA : THE EFFECT OF SUBSTITUTING KCl FOR NaCl IN ARTIFICIAL SEA WATER

The P.D. across the protoplasm of Valonia macrophysa has been studied while the cells were exposed to artificial solutions resembling sea water in which the concentration of KCl was varied from 0 to 0.500 mol per liter. The P.D. across the protoplasm is decreased by lowering and increased by raising the concentration of KCl in the external solution. Changes in P.D. with time when the cell is treated with KCl-rich sea water resemble those observed with cells exposed to Valonia sap. Varying the reaction of natural sea water from pH 5 to pH 10 has no appreciable effect on the P.D. across Valonia protoplasm. Similarly, varying the pH of KCl-rich sea water within these limits does not alter the height of the first maximum in the P.D.-time curve. The subsequent behavior of the P.D., however, is considerably affected by the pH of the KCl-rich sea water. These changes in the shape of the P.D.-time curve have been interpreted as indicating that potassium enters Valonia protoplasm more rapidly from alkaline than from acidified KCl-rich sea water. This conclusion is discussed in relation to certain theories which have been proposed to explain the accumulation of KCl in Valonia sap. The initial rise in P.D. when a Valonia cell is transferred from natural sea water to KCl-rich sea water has been correlated with the concentrations of KCl in the sea waters. It is assumed that the observed P.D. change represents a diffusion potential in the external surface layer of the protoplasm, where the relative mobilities of ions may be supposed to differ greatly from their values in water. Starting with either Planck''s or Henderson''s formula, an equation has been derived which expresses satisfactorily the observed relationship between P.D. change and concentration of KCl. The constants of this equation are interpreted as the relative mobilities of K⁺, Na⁺, and Cl^- in the outer surface layer of the protoplasm. The apparent relative mobility of K⁺ has been calculated by inserting in this equation the values for the relative mobilities of Na⁺ (0.20) and Cl^- (1.00) determined from earlier measurements of concentration effect with natural sea water. The average value for the relative mobility of K⁺ is found to be about 20. The relative mobility may vary considerably among different individual cells, and sometimes also in the same individual under different conditions. Calculation of the observed P.D. changes as phase-boundary potentials proved unsatisfactory.     

The effect of ionic strength on the kinetics of fluoride binding to cytochrome c peroxidase.

The kinetics of the reaction between cytochrome c peroxidase and fluoride was investigated as a function of ionic strength over the pH range 4 to 8. The ionic strength was varied between 0.01 and 0.10 m. At 0.01 m ionic strength, the reaction rates were determined between pH 2.7 and 9.2. A consideration of the ionic strength and pH dependence of the association rate constant for the fluoride-cytochrome c peroxidase reaction leads to the conclusion that hydrofluoric acid is the only significant reactive form of the ligand between pH 2.5 and 8. Above pH 8, binding of fluoride anion contributes to the apparent association rate even though the pH-independent rate constant for fluoride anion binding is more than 3 × 10⁵ times smaller than the rate constant for hydrofluoric acid binding.     

Purification and partial characterization of citrate synthase from Phaseolus vulgaris mitochondria

Citrate synthase (E.C. 4.1.3.7) has been isolated from bean mitochondria by an improved procedure. The purified enzyme had a specific activity of 50. In most respects (e.g. sedimentation constant, K_ms, pH sensitivity and ionic strength inhibition) the enzyme is similar to that prepared from mammalian sources. The feature distinguishing the plant enzyme from the others was its inhibition by several sulfhydryl reagents. The substrates conferred either complete protection (acetyl coenzyme A) or partial protection (oxalacetic acid) against the inhibition. Dithiothreitol (DTT) was capable of partially reversing the inhibition. The efficacy of DTT varied with the sulfhydryl reagent and was inversely related to the period of incubation of the enzyme with the reagent.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.