THE INFLUENCE OF pH UPON THE CONCENTRATION POTENTIALS ACROSS THE SKIN OF THE FROG

The production of concentration P.D.''s across the skin of the frog is very intimately related to the pH of the applied solutions. On the alkaline side of an isoelectric point the dilute solution is electropositive; on the acid side this solution becomes electronegative. When the pH is suddenly lowered from a value more alkaline than this isoelectric point to one considerably more acid the change in polarity may occur within a few seconds. The effect is reversible. When a series of unbuffered solutions at different pH values are applied reversal curves may be obtained. When the concentration gradient is .1 N-.001 N KCl the reversal points lie between pH 4.1 and 4.8. When studied in acetate buffers this electromotive reversal is found to be closely correlated with the electrical charge upon the membrane, as determined by electroendosmosis through it. Reversal occurs between pH 4.9 and 5.2. It is concluded that the electromotive behavior of this material is controlled by some ampholyte, or group of ampholytes, within the membrane. This ampholyte is probably a protein. On both sides of their isoelectric point these membranes, in common with protein membranes, behave as if they retarded or prevented the movement through them of ions of the same electrical sign as they themselves bear, while permitting the movement of ions of the opposite sign. It is suggested that this correlation arises because of electrostatic effects between the charged surfaces and ions in the solution.     

AMPHOTERIC COLLOIDS : II. VOLUMETRIC ANALYSIS OF ION-PROTEIN COMPOUNDS; THE SIGNIFICANCE OF THE ISOELECTRIC POINT FOR THE PURIFICATION OF AMPHOTERIC COLLOIDS.

1. It is shown by volumetric analysis that on the alkaline side from its isoelectric point gelatin combines with cations only, but not with anions; that on the more acid side from its isoelectric point it combines only with anions but not with cations; and that at the isoelectric point, pH = 4.7, it combines with neither anion nor cation. This confirms our statement made in a previous paper that gelatin can exist only as an anion on the alkaline side from its isoelectric point and only as a cation on the more acid side of its isoelectric point, and practically as neither anion nor cation at the isoelectric point. 2. Since at the isoelectric point gelatin (and probably amphoteric colloids generally) must give off any ion with which it was combined, the simplest method of obtaining amphoteric colloids approximately free from ionogenic impurities would seem to consist in bringing them to the hydrogen ion concentration characteristic of their isoelectric point (i.e., at which they migrate neither to the cathode nor anode of an electric field). 3. It is shown by volumetric analysis that when gelatin is in combination with a monovalent ion (Ag, Br, CNS), the curve representing the amount of ion-gelatin formed is approximately parallel to the curve for swelling, osmotic pressure, and viscosity. This fact proves that the influence of ions upon these properties is determined by the chemical or stoichiometrical and not by the "colloidal" condition of gelatin. 4. The sharp drop of these curves at the isoelectric point finds its explanation in an equal drop of the water solubility of pure gelatin, which is proved by the formation of a precipitate. It is not yet possible to state whether this drop of the solubility is merely due to lack of ionization of the gelatin or also to the formation of an insoluble tautomeric or polymeric compound of gelatin at the isoelectric point. 5. On account of this sudden drop slight changes in the hydrogen ion concentration have a considerably greater chemical and physical effect in the region of the isoelectric point than at some distance from this point. This fact may be of biological significance since a number of amphoteric colloids in the body seem to have their isoelectric point inside the range of the normal variation of the hydrogen ion concentration of blood, lymph, or cell sap. 6. Our experiments show that while a slight change in the hydrogen ion concentration increases the water solubility of gelatin near the isoelectric point, no increase in the solubility can be produced by treating gelatin at the isoelectric point with any other kind of monovalent or polyvalent ion; a fact apparently not in harmony with the adsorption theory of colloids, but in harmony with a chemical conception of proteins.     

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 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 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.     

A PHYSICAL-CHEMICAL DIFFERENCE IN ANTIBODIES AGAINST THE S AND R VARIANTS OF A SINGLE BACTERIAL STRAIN

1. Rabbits were immunized with Bact. typhosum 0 901 S and 0 901 R, over a long period. Homologous and heterologous strains were sensitized with sera obtained from weekly bleedings. Agglutination titer was recorded, and the isoelectric points of the bacteria maximally sensitized were determined. 2. 0 901 S maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering a range of pH 4.8 to 5.5. 3. Strain 0 901 R maximally sensitized with homologous immune serum had isoelectric points which became more alkaline as immunization progressed, covering the range of pH 5.0 to 5.9. 4. Both 0 901 S and 0 901 R maximally sensitized with heterologous serum had isoelectric points lower than when sensitized with homologous serum. 5. The isoelectric points of both forms sensitized with increasing concentrations of homologous immune serum were determined. Increasing concentrations of homologous immune serum shifted the isoelectric point of 0 901 R from less than 2.2 for the unsensitized bacteria progressively to the alkaline side until the maximum values previously mentioned were reached. Increasing concentrations of homologous immune serum conferred upon 0 901 S isoelectric points which became only slightly more alkaline in maximal sensitization. 6. The electrophoretic mobilities of 0 901 S and 0 901 R, in each case maximally sensitized with homologous hyperimmune serum, were found to differ significantly over the whole range of pH studied.     

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates. This revised version was published online in July 2006 with corrections to the Cover Date.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF COLLODION MEMBRANES BY TRIVALENT CATIONS

1. Trivalent cations cause a collodion membrane covered with a protein film to be charged positively while they do not produce such an effect on collodion membranes not possessing a protein film. The same had been found for the reversal of the sign of charge of the membrane by acid. 2. This reversal in the sign of charge of the membrane by trivalent cations occurs on the alkaline side of the isoelectric point of the protein used; while the reversal by acid occurs on the acid side of the isoelectric point. 3. The reversal seems to be due to or to be accompanied in both cases by a chemical change in the protein. The chemical change which occurs when the hydrogen ions reverse the sign of charge of the protein film consists in the formation of a protein-acid salt whereby the H ion becomes part of a complex protein cation; while the chemical change which occurs when trivalent cations reverse the sign of charge of the protein film consists in the formation of an insoluble and therefore sparingly or non-ionizable metal proteinate.     

Influence of mono- and multivalent cations on the electrokinetic properties of Rana pipiens kidney cells.

Changes in the cation composition of the ambient cell environment, at constant ionic strengths, altered the electrokinetic properties of the surface of normal adult Rana pipiens kidney cells in a manner which reflected its valence. the effect was expressed in the changes of the magnitude of the net surface charge, at any pH, and in the shift of the isoelectric point of the cell surface.     

ELECTROKINETIC PHENOMENA. III : THE "ISOELECTRIC POINT" OF NORMAL AND SENSITIZED MAMMALIAN ERYTHROCYTES

A survey of the published electrophoretic mobilities of certain mammalian red cells reveals that the isoelectric points accorded to these cells are the result of equilibria incidental to red cell destruction. The electrophoretic mobilities of normal washed sheep and human cells have now been studied in 0.85 per cent NaCl solutions from about pH 3.6 to 7.4. All measurements were made within 2 minutes of the preparation of the suspension of red cells. In no case was reversal of sign of charge observed under these conditions. Reversal of sign of charge occurred only after sufficient time had elapsed to permit sufficient adsorption of the products of red cell destruction. There is little change in mobility as the pH of the medium is decreased. Reversal of sign of charge does occur in the presence of normal and immune (anti-sheep) rabbit sera. The isoelectric point determined under these conditions does not appear to be connected specifically with the immune body but is perhaps associated with phenomena incidental to red cell destruction and the presence of serum. The characteristic lowering of mobility by amboceptor occurs, however, from pH 4.0 to pH 7.4. The curves of mobility plotted against pH for normal and for immune sera support the viewpoint that the identity of the isoelectric points for normal and sensitized sheep cells is not primarily concerned with the immune reaction. It is most unlikely that an "albumin" or a "globulin" surface covers red cells with a complete protein film. Although serum protein reacts with red cells in acid solutions, this is not demonstrable for gelatin. The lowering of mobility usually ascribed to anti-sheep rabbit serum may also occur, but to a lesser degree, in normal rabbit serum. This diminution of mobility is not, in the first place, associated with sensitization to hemolysis induced by complement. This supports the view that only a very small part of the red cell surface need be changed in order to obtain complete hemolysis in the presence of complement.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Binding of sodium n-dodecyl sulphate and protons to catalase

The binding of sodium n-dodecyl sulphate to catalase has been measured by equilibrium dialysis in the pH range 3.2 to 10.0. On the acid side of the isoelectric point (pH 5.4) the surfactant anions initially bind to cationic sites on the protein and subsequent binding is cooperative. At high pH on the alkaline side of the isoelectric point only cooperative binding is observed. The binding data have been combined with protein titration curves to calculate the Gibbs energies of formation of protein titration curves to calculate the Gibbs energies of formation of protein surfactant proton complexes. Contributions to the Gibbs energies of complex formation by surfactant and protein binding have been estimated. The average Gibbs energies of surfactant binding to specific cationic sites are ca. 28 kJ mol^?1 and for cooperative binding ca. 15 kJ mol^?1.     

AMPHOTERIC COLLOIDS : III. CHEMICAL BASIS OF THE INFLUENCE OF ACID UPON THE PHYSICAL PROPERTIES OF GELATIN.

1. The method of removing the excess of hydrobromic acid after it has had a chance to react chemically with gelatin has permitted us to measure the amount of Br in combination with the gelatin. It is shown that the curves representing the amount of bromine bound by the gelatin are approximately parallel with the curves for the osmotic pressure, the viscosity, and swelling of the gelatin solution. This proves that the curves for osmotic pressure are an unequivocal function of the number of gelatin bromide molecules formed under the influence of the acid. The cc. of 0.01 N Br in combination with 0.25 gm, of gelatin we call the bromine number. 2. The explanation of this influence of the acid on the physical properties of gelatin is based on the fact that gelatin is an amphoteric electrolyte, which at its isoelectric point is but sparingly soluble in water, while its transformation into a salt with a univalent anion like gelatin Br makes it soluble. The curve for the bromine number thus becomes at the same time the numerical expression for the number of gelatin molecules rendered soluble, and hence the curve for osmotic pressure must of necessity be parallel to the curve for the bromine number. 3. Volumetric analysis shows that gelatin treated previously with HBr is free from Br at the isoelectric point as well as on the more alkaline side from the isoelectric point (pH ≧ 4.7) of gelatin. This is in harmony with the fact that gelatin (like any other amphoteric electrolyte) can dissociate on the alkaline side of its isoelectric point only as an anion. On the more acid side from the isoelectric point gelatin is found to be in combination with Br and the Br number rises with the pH. 4. When we titrate gelatin, treated previously with HBr but possessing a pH = 4,7, with NaOH we find that 25 cc. of a 1 per cent solution of isoelectric gelatin require about 5.25 to 5.5 cc. of 0.01 N NaOH for neutralization (with phenolphthalein as an indicator). This value which was found invariably is therefore a constant which we designate as "NaOH (isoelectric)." When we titrate 0.25 gm. of gelatin previously treated with HBr but possessing a pH < 4.7 more than 5.5 cc. of 0.01 N NaOH are required for neutralization. We will designate this value of NaOH as "(NaOH)_n," where n represents the value of pH. If we designate the bromine number for the same pH as "Br_n" then we can show that the following equation is generally true: (NaOH)_n = NaOH (isoelectric) + Br_n. In other words, titration with NaOH of gelatin (previously treated with HBr) and being on the acid side of its isoelectric point results in the neutralization of the pure gelatin (NaOH isoelectric) with NaOH and besides in the neutralization of the HBr in combination with the gelatin. This HBr is set free as soon as through the addition of the NaOH the pH of the gelatin solution becomes equal to 4.7. 5. A comparison between the pH values and the bromine numbers found shows that over 90 per cent of the bromine or HBr found was in our experiments in combination with the gelatin.     

Characterization of an Extracellular Protease from the Insect Pathogen Xenorhabdus luminescens

Xenorhabdus luminescens Hm cultured in gelatin broth produced a single extracellular protease. The protease was purified by a factor of 500 and characterized as a monomeric protein with an approximate molecular weight of 61,000. On the basis of inhibitor studies and its pH optimum, the protease was classified as an alkaline metalloprotease with a pH optimum near 8; the isoelectric point of the enzyme is 4.2 +/- 0.2. The protease may be a major factor in the ecology of X. luminescens, which is carried as a symbiom of some parasitic nematodes.     

Characterisation of ionogenic groups and estimation of the net negative electric charge on the surface of cells using natural pH gradients

The technique of isoelectric focusing has been extended to the study of the cell surface. A few tumour cell types and normal liver cells have been examined and are found to have characteristic isoelectric points. The isoelectric point of a cell, it is shown, provides information about the ionogenic groups present on its surface. The net electric charge borne by cells at their isoelectric points can be used to predict their electrophoretic mobilities in buffers at physiological pH and ionic strengths.     

Numerical calculations of the pH of maximal protein stability. The effect of the sequence composition and three-dimensional structure.

A large number of proteins, found experimentally to have different optimum pH of maximal stability, were studied to reveal the basic principles of their preference for a particular pH. The pH-dependent free energy of folding was modeled numerically as a function of pH as well as the net charge of the protein. The optimum pH was determined in the numerical calculations as the pH of the minimum free energy of folding. The experimental data for the pH of maximal stability (experimental optimum pH) was reproducible (rmsd = 0.73). It was shown that the optimum pH results from two factors - amino acid composition and the organization of the titratable groups with the 3D structure. It was demonstrated that the optimum pH and isoelectric point could be quite different. In many cases, the optimum pH was found at a pH corresponding to a large net charge of the protein. At the same time, there was a tendency for proteins having acidic optimum pHs to have a base/acid ratio smaller than one and vice versa. The correlation between the optimum pH and base/acid ratio is significant if only buried groups are taken into account. It was shown that a protein that provides a favorable electrostatic environment for acids and disfavors the bases tends to have high optimum pH and vice versa.     

The separation of proteins with heteropolyacids

The precipitation of proteins with heteropolyacids has been studied for the purpose of large scale primary purification. A precipitate will form if the pH of the reaction between purified ovalbumin, hemoglobin, trypsin, pepsin, bovine serum albumin, ovomucoid, gelatin or ribonuclease and tungstrophosphoric, tungstosilicic or molybdosilicic acid is close to the isoelectric point of the protein and does not cause the dissociation of the heteropolyacid. Below the isoelctric point, the percent precipitation depends on the conformational changes of the protein. The precipitation of ovalbumin with tungstophosphoric decreases as the ionic strength of the buffer increases and is independent, of the protein concentration. Mixtures of ovalbumin and bovine serum albumin, though having close isoelectric points, can be separated by varying the concentration of the precipitant. The electropositive groups which combine with the tungstophosphoric acid are guanidino, ε-amino and imidazole. No precipitation is given by the α-amino groups. Filtrates of microbial fermentations containing lactase, glucose aerode-hydrogenase, alkaline protease, amyloglucosidase, and transglucosylase have been purified by precipitation with heteropolyacids.     

THE STABILITY OF BACTERIAL SUSPENSIONS : III. AGGLUTINATION IN THE PRESENCE OF PROTEINS,NORMAL SERUM,AND IMMUNE SERUM.

1. The addition of proteins or serum to suspensions of bacteria, (Bacillus typhosus or rabbit septicemia) at different pH widens the acid agglutination zone and shifts the isoelectric point to that of the added substance. 2. The amount of serum required to agglutinate is much less near the acid agglutination point of the organisms. 3. The addition of immune serum prevents the salt from decreasing the cohesive force between the organisms, and agglutination therefore is determined solely by the potential, provided excess immune body is present. Whenever the potential is decreased below 15 millivolts the suspension agglutinates.     

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.     

Inhibitory effect of taurolipids on Clostridium perfringens sialidase

Taurolipids A and B, which are detergent-type compounds isolated from protozoan Tetrahymena cells, were demonstrated to inhibit strongly the activity of Clostridium perfringens sialidase. On addition of 280 pmol of taurolipid B to 20 mU of the enzyme, the sialidase activity was decreased to 7% of the original activity at pH 5.1 as the optimum pH. The inhibition was non-competitive. Effective inhibition was observed at the acidic region from the isoelectric point of the sialidase, and at a low ionic strength. Both the long chain acyl and sulfonic acid groups of taurolipids were required for the inhibition of the sialidase activity. A mechanism is postulated for the inhibition.