Quantitative measurements on the acid taste and their bearing on the nature of the nerve receptor

Summary Quantitative determinations of the threshold concentrations for sourness of about 40 organic acids have been determined with an accuracy of about 6 per cent. Hydrogen ion concentrations have been obtained by use of the cell Hg/HgCl/KCl sat./organic acid/H₂. It is assumed that only undissociated acid molecules can cross the phase boundary between the aqueous solution and the nerve receptor, and that the stimulation of the sensation of sourness is due solely to hydrogen ion. All acids tasting equally sour should have the same pH within the nerve receptor. From the experimental data, relative concentration gradients for the nonionized acids have been calculated. It is shown that the addition of polar groups, such as OH, Cl, C=C, C=O, COOH, NH₂ to the organic acid make it from 20 to 500 times more difficult for the organic acid to penetrate into the nerve receptor. On the other hand, lengthening the carbon chain by addition of CH₂ groups makes penetration easier. The phenomena can be largely explained on the hypothesis that these organic acids are adsorbed into a tissue which is essentially like fat rather than like protein.     

The nature of the nerve receptor for the acid taste as indicated by the absorption of organic acids by fats and proteins

Summary Distribution coefficients of thirteen organic acids between olive oil and water have been measured. The concentration in the water phase was about.02N. The acids were chosen so as to bring out the influence of certain polar groups, OH, CO, SO₃H, NH₂ and C=C. The effects of these groups are clearly marked, and are in general in the same direction and of the same order of magnitude as their effects on the concentration gradients of the same undissociated acids between water and the nerve receptor in the taste experiments ofTaylor, Farthing andBerman. Specific effects are noted for the NH₂ and the phenyl groups.Measurements have been made of the hydrogen ion concentration of gelatine to which these acids were added. A family of curves was obtained in which the arrangement is determined by the dissociation constant of the acid. The various polar groups affect the position of the curves only so far as they affect the dissociation constants. The results do not parallel the results of the experiments on taste. It is concluded that the nerve ending which the acid penetrates and stimulates to give the sensation of sourness is essentially like a fat (olive oil) rather than like a protein (gelatine) in character.Submitted for publication December 13, 1929.It is a pleasure to acknowledge the aid of MrLindsay Helmholz, a senior student in chemistry of the Johns Hopkins University. He has ably assisted in the experimental part of this investigation.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS

1. Exposure of unfertilized starfish eggs to dilute solutions of weak acids (fatty acids, benzoic and carbonic acids) in isotonic balanced salt solution causes complete activation with the proper durations of exposure. For each acid the rate of activation (reciprocal of optimum duration) varies with concentration and temperature; at a given temperature and within a considerable range of concentrations (e.g. 0.00075 to 0.004 M for butyric acid), this rate is approximately proportional to concentration. We may thus speak of a molecular rate of action characteristic of each acid. 2. In general the molecular rate of action increases with the dissociation constant and surface activity of the acids. In the fatty acid series (up to caproic), formic acid has the most rapid effect, acting about four times as rapidly as acetic; for the other acids the order is: acetic = propionic ≦ butyric < valeric < caproic. Carbonic acid acts qualitatively like the fatty acids, but its molecular rate of action is only about one-fourteenth that of acetic acid. 3. Hydrochloric and lactic acids are relatively ineffective as activating agents, apparently because of difficulty of penetration. Lactic acid is decidedly the more effective. The action of both acids is only slightly modified by dissolving in pure (isotonic NaCl and CaCl₂) instead of in balanced salt solution. 4. The rate of action of acetic acid, in concentrations of 0.002 M to 0.004 M is increased (by 10 to 20 per cent) by adding Na-acetate (0.002 to 0.016) to the solution. The degree of acceleration is closely proportional to the estimated increase in undissociated acetic acid molecules. Activation thus appears to be an effect of the undissociated acid molecules in the external solution and not of the ions. Acetate anions and H ions acting by themselves, in concentrations much higher than those of the solutions used, have no activating effect. The indications are that the undissociated molecules penetrate rapidly, the ions slowly. Having penetrated, the molecules dissociate inside the egg, yielding the ions of the acid. 5. When the rate of activation is slow, as in 0.001 M acetic acid, the addition of Na-acetate (0,008 M to 0.016 M) has a retarding effect, referable apparently to the gradual penetration of acetate ions to the site of the activation reaction with consequent depression of dissociation. 6. An estimate of the C_H of those solutions (of the different activating acids) which activate the egg at the same rate indicates that their H ion concentrations are approximately equal. On the assumptions that only the undissociated molecules penetrate readily, and that the conditions of dissociation are similar inside and outside the egg, this result indicates (especially when the differences in adsorption of the acids are considered) that the rate of activation is determined by the C_H at the site of the activation reaction within the egg.     

Effects of organic acids on ion uptake and retention in barley roots

Effects of several organic acids on ion uptake and retention and on respiration in barley roots having low and high KCl contents were assayed by measurements of K⁺, Na⁺, Ca²⁺, Cl⁻, and oxygen uptake. Organic acids with high pK_a values increase the permeability of roots to ions and decrease respiration when present in sufficient concentrations at pH 5 but have no inhibitory effects at pH 7. Absence of respiratory inhibition in short times and at lower organic acid concentrations, under conditions that immediately produce a permeability increase, indicate that the permeability change is not a result of respiratory inhibition. Effects of formate, acetate, propionate, and glutarate are attributed to entry of undissociated acid molecules into the effective membranes. Lack of a permeability increase with succinate, which has lower distribution coefficients to lipid solvents than do the aliphatic acids, can be explained by failure of sufficient amounts of the hydrophilic succinic acid molecules to penetrate the membranes involved. These experiments suggest that undissociated acid in root membranes can increase permeability of the roots.     

COMPARISON OF SOURNESS OF ORGANIC ACID ANIONS AT EQUAL pH AND EQUAL TITRATABLE ACIDITY

The sourness of six organic acids was compared by pair tests in binary acid solutions of equal pH and equal potential hydrogen ion concentration. Monoprotic lactic acid was more sour than three of the four acids against which it was tasted, while triprotic citric acid was less sour than the four other acids against which it was compared. Of the diprotic acid pairs which were evaluated, only one significant difference in sourness was observed; succinic acid was more sour than malic acid. No relationship between buffer capacity or hydrophobicity and intensity of sourness was observed.     

The sour taste of acids. The hydrogen ion and the undissociated acid as sour agents

In the first of two experiments, the sourness of aqueous solutionsof HCl and of several carboxylic acids was measured by meansof a fitter-paper method. Perceived sourness of the carboxylicacids was positively correlated with the dissociation constantK_al. In contrast to HCl, the H⁺ concentration in carboxylicacids appeared not to be the only factor in eliciting sourness.Decreasing pH by buffering of the carboxylic acids with theirsodium salts did not lower perceived sourness. The rank-orderof the acids, according to the amount of NaOH needed for titration,depends on the pH to which it is titrated. The rank-order ofpH 4.4 reasonably fits the sourness rank-order. In the secondexperiment, adaptation and cross-adaptation of HCl, tartaric-lactic-,and acetic acid were measured. Neither self- nor cross-adaptationwas observed in the case of HCl, whereas with the carboxylicacids self- and mutual cross-adaptation did occur. The presentresults suggest that HCl-sourness and the sourness of carboxylicacids are elicited by different receptor processes.     

THE ACTIVATION OF STARFISH EGGS BY ACIDS : II. THE ACTION OF SUBSTITUTED BENZOIC ACIDS AND OF BENZOIC AND SALICYLIC ACIDS AS INFLUENCED BY THEIR SALTS.

1. Comparison of the rates of activation of unfertilized starfish eggs in pure solutions of a variety of parthenogenetically effective organic acids (fatty acids, carbonic acid, benzoic and salicylic acids, chloro- and nitrobenzoic acids) shows that solutions which activate the eggs at the same rate, although widely different in molecular concentration, tend to be closely similar in C_H. The dissociation constants of these acids range from 3.2 x 10^–7 to 1.32 x 10^–3. 2. In the case of each of the fourteen acids showing parthenogenetic action the rate of activation (within the favorable range of concentration) proved nearly proportional to the concentration of acid. The estimated C_H of solutions exhibiting an optimum action with exposures of 10 minutes (at 20°) lay typically between 1.1 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.96), and in most cases between 1.6 x 10^–4 M and 2.1 x 10^–4 M (pH = 3.7–3.8). Formic acid (C_H = 4.2 x 10^–4 M) and o-chlorobenzoic acid (C_H = 3.5 x 10^–4 M) are exceptions; o-nitrobenzoic acid is ineffective, apparently because of slow penetration. 3. Activation is not dependent on the penetration of H ions into the egg from without, as is shown by the effects following the addition of its Na salt to the solution of the activating acid (acetic, benzoic, salicylic). The rate of activation is increased by such addition, to a degree indicating that the parthenogenetically effective component of the external solution is the undissociated free acid. Apparently the undissociated molecules alone penetrate the egg freely. It is assumed that, having penetrated, they dissociate in the interior of the egg, furnishing there the H ions which effect activation. 4. Attention is drawn to certain parallels between the physiological conditions controlling activation in the starfish egg and in the vertebrate respiratory center.     

THE RELATIONSHIP BETWEEN CHEMICAL STRUCTURE AND THE RESPONSE OF BLOWFLIES TO TARSAL STIMULATION BY ALIPHATIC ACIDS

Using the technique of proboscis extension in antennectomized-labellectomized flies, the rejection thresholds of Phormia regina for 18 fatty acids and one mineral acid have been determined. The conclusions reached on the basis of these data may be summarized in the following terms: Tarsal stimulation by acids involves the summation of components from at least two sources. Of these the hydrogen ion is the most important. The other major factor is probably the anion rather than the undissociated acid. The stimulating power of the anions (or free acid molecules) increases with increasing chain length in both the mono- and dicarboxylic series, but the rate of increase decreases as the series is ascended. Acids containing 6 or more carbon atoms are not sufficiently soluble in 0.1 M sucrose to reach the threshold of rejection. Substitution of —H in the acyl grouping by —Cl, —OH, =O or —COOH, the presence of a C=C bond, or a shift from the trans- to the cis- configuration all diminish the effectiveness of the anion (or free acid). But since such alterations also augment the degree of dissociation and consequently the concentration of hydrogen ions, the net result is ordinarily a lowering of threshold in terms of the molar concentration required for rejection.     

Astringency of Organic Acids is Related to pH

Astringency and sourness of lactic, acetic and citric acids,each adjusted to pH 3, 5 and 7, were evaluated in two experiments,one starting at equal concentrations in wt/vol before neutralizationand the second starting at equal molarity. Astringency and sournessdecreased with increasing pH. However, acids were differentiallysour at equal pH, consistent with previous findings. In contrast,the tactile attributes associated with astringency (drying,roughing of oral tissues and puckery/tightening sensations)were similar across acids; pH was the major influence on astringency.Strong dependence on pH suggests that astringency of these acidsis a direct result of their acidic properties, and not solelydue to the hydrogen bonding mechanisms previously suggestedas an explanation of astringency in tannin interactions withsalivary proteins. Chem. Senses 21: 397–403, 1996.     

Comparison of the effects of concentration, pH and anion species on astringency and sourness of organic acids

The separate effects of concentration, pH and anion species on intensity of sourness and astringency of organic acids were evaluated. Judges rated sourness and astringency intensity of lactic, malic, tartaric and citric acid solutions at three levels of constant pH varying in normality and at three levels of constant concentration varying in pH. To assess the comparative sourness and astringency of the organic acid anions of study, binary acid solutions matched in pH and titratable acidity were also rated. As pH was decreased in equinormal solutions, both sourness and astringency increased significantly (P < 0.001). By contrast, as the normality of the equi-pH solutions was increased, only sourness demonstrated significant increases (P < 0.001) while astringency remained constant or decreased slightly. At the lowest normality tested, all solutions were more astringent than sour (P < 0.05). Although lactic acid was found to be significantly more sour than citric acid (P < 0.05), no other sourness or astringency differences among the organic acid anions were noted. This study demonstrates for the first time that astringency elicited by acids is a function of pH and not concentration or anion species, and confirms that sourness is independently influenced by concentration, pH and anion species of the acid.      

Theoretical results for chemotactic response and drift of E. coli in a weak attractant gradient

The bacterium Escherichia coli (E. coli) moves in its natural environment in a series of straight runs, interrupted by tumbles which cause change of direction. It performs chemotaxis towards chemo-attractants by extending the duration of runs in the direction of the source. When there is a spatial gradient in the attractant concentration, this bias produces a drift velocity directed towards its source, whereas in a uniform concentration, E. coli adapts, almost perfectly in case of methyl aspartate. Recently, microfluidic experiments have measured the drift velocity of E. coli in precisely controlled attractant gradients, but no general theoretical expression for the same exists. With this motivation, we study an analytically soluble model here, based on the Barkai-Leibler model, originally introduced to explain the perfect adaptation. Rigorous mathematical expressions are obtained for the chemotactic response function and the drift velocity in the limit of weak gradients and under the assumption of completely random tumbles. The theoretical predictions compare favorably with experimental results, especially at high concentrations. We further show that the signal transduction network weakens the dependence of the drift on concentration, thus enhancing the range of sensitivity.     

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V_max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.     

Uptake and Release of Abscisic Acid by Isolated Photoautotrophic Mesophyll Cells, Depending on pH Gradients

Uptake and release of abscisic acid (AbA) by isolated mesophyll cells of Papaver somniferum is characterized by the following observations: (a) Uptake rate is a linear function of the external AbA concentration in the range from 10⁻⁶ to 5 × 10⁻⁵ molar, and decreases with increasing pH. At any pH, uptake rate is linearly related to the concentration of undissociated abscisic acid, calculated from the pK = 4.7 according to the Henderson-Hasselbalch equation. At low external pH (5.0), AbA accumulation in the cells is about 10-fold. (b) Uptake of AbA is completely inhibited by salts such as KNO₂ or sodium acetate, which decrease the pH gradient between medium and cells. KCN or m-chlorocarbonylcyanide phenylhydrazone inhibits AbA uptake only after longer incubation periods (20-40 minutes). (c) Uptake rate as well as equilibrium concentration is significantly higher in light than in darkness. (d) At low external pH, release of AbA from preloaded cells is strongly stimulated by KNO₂. It is concluded that AbA is distributed between leaf cells and free space according to pH gradients, with the undissociated abscisic acid being the main penetrating species. Uptake and release occur via diffusion, without participation of a carrier.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Effect of pH on Penetration of Naphthaleneacetic Acid and Naphthaleneacetamide Through Isolated Pear Leaf Cuticle

Penetration of naphthaleneacetic acid through enzymatically isolated upper pear (Pyrus communis L. cv. Bartlett) leaf cuticle increased as the donor pH was decreased. Naphthaleneacetamide penetration was not influenced by donor pH. The effect of pH on naphthaleneacetic acid penetration was reversible. Higher receiver (simulated leaf interior) pH favored penetration of naphthaleneacetic acid. Changes in the degree of dissociation, and hence polarity, as controlled by hydrogen ion concentration was the prime factor in the response of naphthaleneacetic acid to pH. At pH values lower than the pK (4.2 for naphthaleneacetic acid), the molecule was primarily undissociated, lipophilic, and penetrated into the cuticle; whereas, at pH values above the pK naphthaleneacetic acid was ionized, hydrophilic, and penetrated the cuticle with difficulty or not at all. Data presented are consistent with the hypothesis that naphthaleneacetic acid and naphthaleneacetamide penetration through the cuticle takes place by diffusion.     

CELL PENETRATION BY ACIDS : VI. THE CHLOROACETIC ACIDS.

Measurements of the penetration of tissue from Chromodoris zebra are believed to show that a determining factor in penetration involves the establishment of a critical pH (near 3.5) in relation to superficial cell proteins. The rapidity with which this state is produced depends upon acid strength, and upon some property of the acid influencing the speed of absorption; hence it is necessary to compare acids within groups of chemical relationship. The actual speed of penetration observed with any acid is dependent upon two influences: preliminary chemical combination with the outer protoplasm, followed by diffusion. The variation of the temperature coefficient of penetration velocity with the concentration of acid, and the effect of size (age) of individual providing the tissue sample agree in demonstrating the significant part played by diffusion. In comparing different acids, however, their mode of chemical union with the protoplasm determines the general order of penetrating ability.     

THE PENETRATION OF CO2 INTO LIVING PROTOPLASM

The experiments indicate that little or no CO₂ enters normal cells of Valonia except in the form of undissociated molecules. Whenever the interior of a cell is more acid than the surrounding medium (excess base being the same in both) we may expect that at equilibrium the internal concentration of total CO₂ will be less than the external.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

Astringent Subqualities in Acids

Astringency, astringent subqualities (drying, roughing and puckering)and sourness were compared among six acids: hydrochloric, lactic,citric, acetic, fumaric and malic acids. The attribute profilesof organic acids were similar to each other but different fromhydrochloric acid, the only inorganic acid, which was the mostastringent and the least sour. In a second experiment, two inorganicacids (hydrochloric and phosphoric) and two organic acids (citricand malic) were tested at three concentration levels. At approximatelyequal levels of overall sensory impact, the inorganic acidswere alike in astringency and sourness, receiving higher ratingsfor roughing and drying, and lower ratings for sourness thanthe organic acids. Interactions with concentration (differencesin psychophysical functions) for the subquality of drying werenoted, in addition to the differences in the astringent subqualitiesof roughing and drying seen across acids in both experiments.The higher level of astringency for inorganic acids suggeststhat the current model for tannin binding to salivary proteinsas an explanation of astringency needs to be extended to includea direct pH-dependent effect. Chem. Senses 20: 593–600,1995     

The Bactericidal Action of Low Molecular Weight Compounds on Mycobacterium tuberculosis

Summary: Lower members of the carboxylic fatty acid series, hydroxy acids, alcohols, amides, ureas, urethanes and some other low molecular weight compounds have been tested for bactericidal action on Mycobacterium tuberculosis H37 Rv and BCG. The rate of bactericidal action of acetic acid is shown to be dependent on the concentration of undissociated acid. The bactericidal activity of normal fatty acids increases with increase in the length of the carbon chain. A comparison of the times required by equimolar solutions to sterilize a culture shows that maximum activity is reached at undecylic acid (C₁₁). The bactericidal activity of corresponding acids, hydroxy acids and alcohols is compared.