Proton movements across the membranes of sarcoplasmic reticulum during the uptake of calcium ions

A burst of proton ejection was observed during the initial steps of Ca²⁺ uptake by sarcoplasmic reticulum vesicles. The initial rate of this proton ejection is considerably higher than the initial rate of Ca²⁺ uptake, and is independent of the amount of accumulated Ca²⁺. The ejection of protons is a transmembrane event, since it is dissipated by the ionophore X-537A, and does not occur when the ionophore is added before the initiation of the transport of Ca²⁺. The low proton permeability of the membranes is largely increased by X-537A. The studies of facilitated diffusion of protons in the presence of the ionophore permitted the estimation of the pH within the vesicles. A fast alkalinization occurs within the vesicles during the initial steps of Ca²⁺ uptake, as revealed by sequestered bromothymol blue. The change in absorbance of this dye corresponds to a change of 0.15 pH unit within the vesicles, and a maximal transmembrane ΔpH of about 0.5 may build up. Since such a gradient may not account energetically for the transmembrane gradients of Ca²⁺, I suggest that a transmembrane electrical potential may develop as a consequence of proton ejection.     

The effect of intracellular pH on the rate of hexose uptake in Chlorella.

The rate of hexose uptake by Chlorella is reduced by uncouplers such as carbonyl cyanide p-trifluoromethoxyphenyl hydrazone or dinitrophenol even before concentration equilibrium is reached. The addition of uncouplers changes the membrane potential and the intracellular pH. The membrane potential does not influence the initial velocity of net sugar uptake, whereas manipulation of the cell pH by means of dimethyloxazolidinedione or by butyric acid uncovered a dramatic influence of cell pH on the rate of hexose uptake: at pH values of 7.5--6.8 maximal rate of uptake is observed but at more acid pH a strong inhibition takes place with virtually total blockage of uptake at pH 6.1. The decrease of cell pH to 6.1 in the presence of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone could therefore account for the decrease in hexose transport rate. It was shown that the intracellular pH as such determines the rate of uptake and not the pH difference between inside and outside; the transport rate did not correlate with delta pH.     

Probing of the substrate binding domain of lactose permease by a proton pulse

The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.     

Postillumination adenosine triphosphate synthesis in Rhodospirillum rubrum chromatophores. I. Conditions for maximal yields.

The very low level of postillumination ATP synthesis in chromatophores was markedly stimulated when permeant anions (thiocyanate or perchlorate) or permeant cations (potassium in the presence of valinomycin) were added to the light stage. Although these compounds stimulated also light-induced proton uptake in chromatophores the pH dependence of both photoreactions was different. Proton uptake peaked at pH 6.5 while the amount of postillumination ATP was maximal when the light stage was carried out around pH 7.7. The increased yield of ATP at the more alkaline pH could not be explained by a slower decay of the high energy state at this pH, since the decay rate was faster at pH 7.7 than at pH 6.5. The proton concentration gradient which is maintained across the chromatophore membrane in the light was also found to increase when the external pH was raised from 6.0 to 8.0. Only a minimal amount of postillumination ATP was formed when this gradient was below 2.1 pH units, but above this value the ATP yield rose steeply as a function of the increasing pH gradient. In light of these results it is suggested that in order to obtain a high yield of postillumination ATP synthesis in chromatophores two conditions are required: the particles have to be loaded with a sufficient number of protons and a light-induced pH gradient above a certain threshold value has to be maintained across their membrane. The low yield of postillumination ATP in chromatophores and the increase obtained by adding permeating ions, is thus explained by similar variations in the extent of the pH gradient, which exceeded the threshold value only in the presence of the permeating ions.     

The determination of the membrane ptoential of Chlorella vulgaris. Evidence for electrogenic sugar transport.

From data on the accumulation of tetraphenylphosphonium within Chlorella vulgaris cells, it can be estimated that these cells possess a membrane potential of --120 to --150 mV (inside negative). Under anaerobic conditions as well as in the presence of uncoupling agents the membrane potential drops to about -60 to -80 mV. Nystatin (50 mug/ml) abolishes it almost completely. Since it took more than 1 h before the tetraphenylphosphonium equilibrium was reached, this method could not be used to measure relatively fast transient changes in membrane potential. However, the rate of influx of tetraphenylphosphonium is also directly dependent on membrane potential and can be followed within minutes. Using this phenomenon as an indicator for membrane potential a brief transient depolarisation was detected after the addition of sugars taken up by Chlorella via the proton cotransport system. The depolarisation was absent from cells not induced for sugar uptake and induced cells did not show it with substances not transported, like mannitol. The maximal depolarisation observed amounted to about 70 mV; after 1 min, however, the membrane potential returned to a value about 25 mV less negative than the one before sugars was added. The results demonstrate that sugar uptake in Chlorella is electrogenic. The delta pH plus membrane potential measured for Chlorella completely cover the energy required to explain the 1600-fold accumulation of 6-deoxyglucose experimentally observed.     

The effect of vanadate on proton-sucrose cotransport in ricinus cotyledons

The effects of orthovanadate on the uptake of sucrose by Ricinus cotyledons and on sucrose-coupled proton influx were measured in order to gain insight into the relationship to the plasma membrane proton pump. Vanadate had no effect on short-term sucrose uptake. In longterm experiments (>30 min) sucrose uptake was progressively inhibited, but only at high external sucrose concentrations. Vanadate did not affect proton efflux pumping in the absence of sucrose and neither did it change the initial rate of sucrose-coupled proton influx. However, it enhanced the maximal level of sucrose-induced alkalinization of the medium at all sucrose concentrations tested. This is interpreted as an inhibiting effect of vanadate on the proton pump that recycles protons during sucrose-proton cotransport. The sensitivity towards vanadate indicates that this proton pump is an ATPase. A second proton-translocating system, that is insensitive to vanadate, is postulated to function in the absence of sucrose.     

On the stimulation of the light-induced proton uptake by uncoupling aminoacridine derivatives in spinach chloroplasts.

1. Light-induced proton uptake by spinach chloroplasts is enhanced several-fold by 9-(4-diethylamino-1-methylbutylamino)-6-chloro-2-methoxyacridine (atebrin). This stimulation does not depend on the chlorophyll concentration. The amount of extra protons taken up in the presence of atebrin is determined by the pKa values of atebrin and the pH of the incubation medium. 2. Both the stimulation of the proton uptake and the maximal binding capacity for atebrin is sensitive to uncouplers. However, the ratio of bound to free atebrin does not depend on the presence of uncoupler up to the saturating atebrin concentration. 3. From simultanious kinetic measurements of atebrin fluorescence and proton movement it seems that after binding of the completely protonated atebrin the dye and the protons can move separately. This can also be inferred from the spectral behaviour of atebrin in illuminated chloroplasts. 4. The stimulation of the proton uptake by atebrin does not depend on the presence of salts in the incubation medium. However, the 'saturating' atebrin concentration increases strongly with increasing salt concentration in the medium. 5. It is concluded that the interaction of atebrin and other acridines with energized chloroplasts most likely occurs at the level of the membrane proper. 6. It is proposed that uncoupling by atebrin is a consequence of the creation of a high proton activity at the periphery of the thylakoid membrane, which opposes a proton gradient across the membrane. The uncoupling by atebrin is not of the protonophoric type according to this mechanism.     

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris. Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system.

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule. It was found that in the presence of 1-deoxyglucose a considerable proportion of "carrier" catalyzes the transport of protons without the concomitant transport of sugar. Presumably, the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues. This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons. (b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.     

The Hexose-Proton Cotransport System of Chlorella : pH-Dependent Change in Km Values and Translocation Constants of the Uptake System

The proton concentration in the medium affects the maximal velocity of sugar uptake with a K_m of 0.3 mM (high affinity uptake). By decreasing the proton concentration a decrease in high affinity sugar uptake is observed, in parallel the activity of a low affinity uptake system (K_m of 50 mM) rises. Both systems add up to 100%. The existence of the carrier in two conformational states (protonated and unprotonated) has been proposed therefore, the protonated form with high affinity to 6-deoxyglucose, the unprotonated form with low affinity. A plot of extrapolated V_max values at low substrate concentration versus proton concentration results in a K_m for protons of 0.14 µM, i.e. half-maximal protonation of the carrier is achieved at pH 6.85. The stoichiometry of protons cotransported per 6-deoxyglucose is close to 1 at pH 6.0–6.5. At higher pH values the stoichiometry continuously decreases; at pH 8.0 only one proton is cotransported per four molecules of sugar. Whereas the translocation of the protonated carrier is strictly dependent on sugar this coupling is less strict for the unprotonated form. Therefore at alkaline pH a considerable net efflux of accumulated sugar can occur. The dependence of sugar accumulation on pH has been measured. The decrease in accumulation with higher pH values can quantitatively be explained by the decrease in the amount of protonated carrier. The properties of the unprotonated carrier resemble strikingly the properties of carrier at the inner side of the membrane. The inside pH of Chlorella was measured with the weak acid 5,5-dimethyl-2, 4-oxazolidinedion (DMO). At an outside pH of 6.5 the internal pH was found to be 7.2. To explain the extent of sugar accumulation it has to be assumed that the membrane potential also contributes to active sugar transport in this alga.     

H+/ATP stoichiometry for the gastric (K++H+)-ATPase

Summary The initial rate of ATP-dependent proton uptake by hog gastric vesicles was measured at pH's between 6.1 and 6.9 by measuring the loss of protons from the external space with a glass electrode. The apparent rates of proton loss were corrected for scalar proton production due to ATP hydrolysis. For vesicles in 150mm KCl and pH 6.1, corrected rates of proton uptake and ATP hydrolysis were 639±84 and 619±65 nmol/min×mg protein, respectively, giving an H⁺/ATP ratio of 1.03±0.7. Furthermore, at all pH's tested the ratio of the rate of proton uptake to the rate of ATP hydrolysis was not significantly different than 1.0. No proton uptake (<10 nmol/min×mg protein) was exhibited by vesicles in 150mm NaCl at pH 6.1 despite ATP hydrolysis of 187±46 nmol/min×mg (nonproductive hydrolysis). Comparison of the rates of proton transport and ATP hydrolysis in various mixture of KCl and NaCl showed that the H⁺/ATP stoichiometries were not significantly different than 1.0 at all concentrations of K⁺ greater than 10mm. This fact suggests that the nonproductive rate is vanishingly small at these concentrations, implying that the measured H⁺/ATP stoichiometry is equal to the enzymatic stoichiometry. This result shows that the isolated gastric (K⁺+H⁺)-ATPase is thermodynamically capable of forming the observed proton gradient of the stomach.     

Subunit III-depleted cytochrome c oxidase provides insight into the process of proton uptake by proteins

We review studies of subunit III-depleted cytochrome c oxidase (CcO III (-)) that elucidate the structural basis of steady-state proton uptake from solvent into an internal proton transfer pathway. The removal of subunit III from R. sphaeroides CcO makes proton uptake into the D pathway a rate-determining step, such that measurements of the pH dependence of steady-state O(2) consumption can be used to compare the rate and functional pK(a) of proton uptake by D pathways containing different initial proton acceptors. The removal of subunit III also promotes spontaneous suicide inactivation by CcO, greatly shortening its catalytic lifespan. Because the probability of suicide inactivation is controlled by the rate at which the D pathway delivers protons to the active site, measurements of catalytic lifespan provide a second method to compare the relative efficacy of proton uptake by engineered CcO III (-) forms. These simple experimental systems have been used to explore general questions of proton uptake by proteins, such as the functional value of an initial proton acceptor, whether an initial acceptor must be surface-exposed, which side chains will function as initial proton acceptors and whether multiple acceptors can speed proton uptake.     

Effect of pH on the oxidation-reduction properties of thioredoxins

Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.     

Different proton-sugar stoichiometries for the uptake of glucose analogues by Chlorella vulgaris: Evidence for sugar-dependent proton uptake without concomitant sugar uptake by the proton-sugar symport system

The uptake of hexoses by Chlorella vulgaris is accompanied by the uptake of protons. For 6-deoxyglucose a stoichiometry of one proton taken up per sugar molecule has been measured, whereas for 1-deoxyglucose approximately two protons are taken up per sugar molecule.It was found that in the presence of 1-deoxyglucose a considerable proportion of “carrier” catalyzes the transport of protons without the concomitant transport of sugar. Presumably the binding of sugar initiates the translocation of the carrier-proton-sugar complex, but whereas 1-deoxyglucose can still dissociate from the complex at the external side of the cytoplasmic membrane, the translocation of the carrier-proton complex continues.This conclusion was reached since (a) the composition of the translocated carrier-proton-sugar complex is the same for both sugar. Its formation is a first order reaction with respect to protons.(b) When 6-deoxyglucose, present inside cells, is exchanged for external sugar, the exchange ratio is two to one when the external sugar is 1-deoxyglucose, two molecules of 6-deoxyglucose are lost for each molecule of 1-deoxyglucose entering. This result indicates that during uptake of 1-deoxyglucose statistically only each second carrier molecule appearing at the internal side of the cytoplasmic membrane is carrying sugar.     

Order of proton uptake and release by bacteriorhodopsin at low pH.

The order of proton uptake and release in an aqueous suspension of purple membrane in response to a light flash has been investigated at lowered pH. pH indicator dyes and a flash spectrophotometer were used for the study. At pH 6.6 it was found that the release of protons from the purple membrane precedes uptake, as reported by other investigators. At pH 5.9, 4.9, and 4.1 it was also found that release precedes uptake. These results are not in agreement with those of previous investigators.     

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H⁺ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H⁺/H₂PO₄⁻ stoichiometry of the cotransport is most likely to be 4.     

Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium.

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.     

Mechanism of proline uptake by Chlorella vulgaris

An amino acid uptake system specific for glycine, alanine, serine and proline was induced by glucose in Chlorella vulgaris. The uptake system translocated the zwitterionic form of the amino acid. There was more than 100-fold accumulation which indicated a coupling to metabolic energy. The depolarization of the membrane potential during proline uptake and the sensitivity of its uptake rate to the membrane potential point to coupling with an ion flow. Inhibitors of plasmalemma-bound H⁺-ATPase inhibit proline uptake. These data are interpreted to mean that proline is taken up as a proton symport. In some Chlorella strains the proline-coupled H⁺ uptake could be measured with electrodes, but not in Chlorella vulgaris. There is evidence that the transport of amino acids rapidly stimulates the proton-translocating ATPase of Chlorella vulgaris, so that the proline-coupled proton uptake is immediately neutralized.     

A respiration-dependent primary sodium extrusion system functioning at alkaline pH in the marine bacterium Vibrioalginolyticus

The membrane potential generated at pH 8.5 by K⁺-depleted and Na⁺-loaded $\text{Vibrio}\text{alginolyticus}$ is not collapsed by proton conductors which, instead, induce the accumulation of protons in equilibrium with the membrane potential. The generation of such a membrane potential and the accumulation of protons are specific to Na⁺-loaded cells at alkaline pH and are dependent on respiration. Extrusion of Na⁺ at pH 8.5 occurs in the presence of proton conductors unless respiration is inhibited while it is abolished by proton conductors at acidic pH. The uptake of α-aminoisobutyric acid, which is driven by the Na⁺-electrochemical gradient, is observed even in the presence of proton conductors at pH 8.5 but not at acidic pH. We conclude that a respiration-dependent primary electrogenic Na⁺ extrusion system is functioning at alkaline pH to generate the proton conductor-insensitive membrane potential and Na⁺ chemical gradient.     

Intramolecular proton-transfer reactions in a membrane-bound proton pump: the effect of pH on the peroxy to ferryl transition in cytochrome c oxidase

In the membrane-bound redox-driven proton pump cytochrome c oxidase, electron- and proton-transfer reactions must be coupled, which requires controlled modulation of the kinetic and/or thermodynamic properties of proton-transfer reactions through the membrane-spanning part of the protein. In this study we have investigated proton-transfer reactions through a pathway that is used for the transfer of both substrate and pumped protons in cytochrome c oxidase from Rhodobacter sphaeroides. Specifically, we focus on the formation of the so-called F intermediate, which is rate limited by an internal proton-transfer reaction from a possible branching point in the pathway, at a glutamic-acid residue (E(I-286)), to the binuclear center. We have also studied the reprotonation of E(I-286) from the bulk solution. Evaluation of the data in terms of a model presented in this work gives a rate of internal proton transfer from E(I-286) to the proton acceptor at the catalytic site of 1.1 x 10(4) s(-1). The apparent pK(a) of the donor (E(I-286)), determined from the pH dependence of the F-formation kinetics, was found to be 9.4, while the pK(a) of the proton acceptor at the catalytic site is likely to be > or = 2.5 pH units higher. In the pH range up to pH 10 the proton equilibrium between the bulk solution and E(I-286) was much faster than 10(4) s(-1), while in the pH range above pH 10 the proton uptake from solution is rate limiting for the overall reaction. The apparent second-order rate constant for proton transfer from the bulk solution to E(I-286) is >10(13) M(-1) s(-1), which indicates that the proton uptake is assisted by a local buffer consisting of protonatable residues at the protein surface.     

Succinate uptake and related proton movements in Escherichia coli K12.

1. The apparent Km values for succinate uptake by whole cells of Escherichia coli K12 depend on pH in the range 6.5-7.4.2. Uptake of succinate in lightly buffered medium is accompanied by proton uptake. 3. The apparent Km values for succinate uptake and for succinate-induced proton uptake are similar. 4. Approximately two protons enter the cell with each succinate molecule. 5. The pattern of inhibition of succinate uptake is similar to that of succinate-induced proton uptake. 6. Uptake of fumarate and malate, which share the succinate-transport system, is also accompanied by the uptake of approximately two protons per molecule of fumarate or malate. 7. Uptake of aspartate by the dicarboxylic acid-transport system is accompanied by the uptake of approximatley two protons per molecule of asparatate. 8. It is concluded that uptake of dicarboxylic acids by the dicarboxylic acid-transport system is obligatorily coupled to proton uptake such that succinate, malate and fumarate are taken up in electroneutral form and asparate is taken up in cationic form. 9. These results are consistent with, though they do not definitely prove, the energization of succinate uptake of the deltapH.