The electrochemical proton gradient in Escherichia coli membrane vesicles.

Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.     

Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.     

The active transport of 2-keto-D-gluconate in vesicles prepared from Pseudomonas purida.

The transport of 2-keto-D-gluconate (alpha-D-arabino-2-hexulopyranosonic acid; 2KGA) in vesicles prepared from glucose-grown Pseudomonas putida occurs by a saturable process with a Km of 110.0 +/- 2.9 microM and a Vmax. of 0.55 +/- 0.04 nmol X min-1 X (mg of protein)-1. The provision of phenazine methosulphate/ascorbate or L-malate leads to an accumulation of intravescular 2KGA, a decrease in the Km value to 50 +/- 2.1 microM and 35 +/- 2.9 microM respectively and no change in the Vmax. In the presence of electron donors the transport of 2KGA is inhibited by the respiratory poisons antimycin A, rotenone and the uncoupler 2,4-dinitrophenol. 2KGA transport is also competitively inhibited by 4-deoxy-4-fluoro-2-keto- or 3-deoxy-3-fluoro-2-keto-D-gluconate with Ki values of 50 microM and 160 microM respectively. The carrier system for 2KGA is repressed in vesicles from cells grown on succinate. Such vesicles transport 2KGA by non-specific physical diffusion with a Km value of infinity in the absence or presence of electron donors. Vesicles from glucose or succinate grown cells, in the presence of phenazine methosulphate/ascorbate at pH 6.6, generate a proton-motive force (delta p) of approx. 140 mV. The delta p, composed of proton gradient (delta pH) and a membrane potential (delta psi), is collapsed in the presence of dinitrophenol. Based on the results obtained with valinomycin, nigericin and carbonyl cyanide m-chlorophenylhydrazone, the active transport of 2KGA at pH 6.6 is coupled predominately to the delta pH component of delta p.     

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.     

Electrochemical proton gradient in Micrococcus lysodeikticus cells and membrane vesicles.

Using the distribution of weak acids to measure the pH gradient (delta pH; interior alkaline) and the distribution of the lipophilic cation [3H]tetraphenylphosphonium+ to monitor the membrane potential (delta psi; interior negative), we studied the electrochemical gradient or protons (delta mu- H+) across the membrane of Micrococcus lysodeikticus cells and plasma membrane vesicles. With reduced phenazine methosulfate as electron donor, intact cells exhibited a relatively constant delta mu- H+ (interior negative and alkaline) of -193 mV to -223 mV from pH 5.5 to pH 8.5. On the other hand, in membrane vesicles under the same conditions, delta mu- H+ decreased from a maximum value of -166 mV at pH 5.5 to -107 mV at pH 8.0 and above. This difference is related to a differential effect of external pH on the components of delta mu- H+. In intact cells, delta pH decreased from about -86 mV (i.e., 1.4 units) at pH 5.5 to zero at pH 7.8 and above, and the decreases in delta pH was accompanied by a reciprocal increase in delta psi from -110 mV at pH 5.5 to -211 mV at pH 8.0 and above. In membrane vesicles, the decrease in delta pH with increasing external pH was similar to that described for intact cells; however, delta psi increased from -82 mV at pH 5.5 to only -107 mV at pH 8.0 and above.     

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase.

Measurements were made of the difference in the electrochemical potential of protons (delta-mu H+) across the membrane of vesicles restituted from the ATPase complex (TF0.F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (delta psi) and pH difference across the membrane (delta pH), respectively. In the presence of Tris buffer the maximal delta psi ans no delta pH were produced, while in the presence of the permeant anion NO-3 the maximal delta pH and a low delta psi were produced by the addition of ATP. When thATP concentration was 0.24 mm, the delta psi was 140-150 mV (positive inside) in Tris buffer, and the delta pH was 2.9-3.5 units (acidic inside) in the presence of NO-3. Addition of a saturating amount of ATP produced somewhat larger delta psi and delta pH values, and the delta -muH+attained was about 310mV. By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4-5 during ATP hydrolysis. The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.     

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out membrane vesicles. Evidence for the development of a positive interior membrane potential

Previous observations on the effects of permeant anions on ATP-dependent calcium transport in inside-out vesicles prepared from human erythrocytes suggested that the calcium pump is electrogenic, generating a positive interior membrane potential. The present work demonstrates the development of a positive interior membrane potential across inside-out vesicle membranes during calcium transport in the absence of permeant anions. Several membrane potential probes, 1-anilino-8-naphthalenesulfonate, 3,3'-dipropylthiodicarbocyanine iodide, and an electron paramagnetic resonant triphenylphosphonium derivative, provide qualitative evidence for the development of a membrane potential. Moreover, a number of parallels are observed between the changes in the membrane potential measured by the probes and calcium transport. These include enhancement by calmodulin, time course of change, similar kinetic properties, and the requirement for intact vesicle membranes. Quantitative measurements of the membrane potential shows a positive interior membrane potential of 26-37 mV using radiolabeled permeant anion distribution and 38-57 mV using 3,3'-dipropylthiodicarbocyanine iodide fluorescence changes. These membrane potentials are of a similar magnitude to those reported for the sarcoplasmic reticulum calcium pump (Zimniak, P., and Racker, E. (1978). J. Biol. Chem. 253, 4631-4637).     

Cyanine dye as monitor of membrane potentials in Escherichia coli cells and membrane vesicles.

The fluorescence response of a positively charged cyanine dye: 3,3'-dimethylindodicarbocyanine iodide can be specifically related to the generation in Escherichia coli cells and E. coli membrane vesicles of an electrical membrane potential induced either by substrate oxidation or by an artificially imposed potassium diffusion gradient. The energy-dependent quenching of the dye fluorescence correlates well with the known effect on delta phi of: oxidation of various energy sources, external pH and solute accumulation. Thus, in the vesicles, the fluorescence quenching of the dye increases from succinate to D-lactate, to ascorbate/phenazine methosulfate and parallels the increasing ability of these electron donors to generate a delta phi. In the vesicles, delta phi is only weakly dependent on external pH, whereas in the cells, delta phi increases with increasing external pH. Lactose accumulation in the vesicles results in the partial utilization of delta phi. A calibration of the dye fluorescence in terms of delta phi has been determined using valinomycin-induced potassium diffusion potential.     

Purification and characterization of Ca2+/H+ antiporter from Bacillus subtilis

Ca2+ was accumulated in inside-out membrane vesicles of Bacillus subtilis when NADH was used as an energy source. A delta pH (acid interior) could also drive Ca2+ accumulation in the membrane vesicles and the accumulation was inhibited by carbonylcyanide p-trifluoromethoxyphenylhydrazone and nigericin plus K+. These results indicate the presence of a Ca2+/H+ antiporter (exchanger) in this organism. The antiporter was isolated and purified to homogeneity from the membrane proteins by chromatography on hydroxyapatite, diethylaminoethyl(DEAE)-Toyopearl 650 M and butyl-Toyopearl 650 M. The purified antiporter has a molecular mass of about 45 000 daltons and an isoelectric point of 5.0. The fluorescence quenching of a cyanine dye (3,3'-dipropylthiodicarbocyanine iodide [diS-C3-(5)] during Ca2+ accumulation in proteoliposomes by the purified antiporter showed the generation of a membrane potential (interior negative) suggesting a H+/Ca2+ stoichiometry above 2 in the transport. This was also supported by the result that the K+-diffusion potential, interior positive, stimulated the Ca2+ uptake in the presence of a delta pH. The apparent Km for Ca2+ of the antiporter was about 40 microM and La3+ inhibited the transport. Amino acid analysis of the purified antiporter indicated the presence of large amounts of glutamic and aspartic acids and small amounts of histidine, lysine and arginine. This is consistent with the low isoelectric point (about 5.0) of the protein.     

Determination of membrane potential with lipophilic cations: correction of probe binding

The binding of lipophilic ions to the membrane of envelope vesicles from Halobacterium halobium was examined in the absence and presence of membrane potential. The lipophilic ions used constitute a homologous series of (Phe)₃-P⁺-(CH₂)_n-CH₃ (n = 0–4) and tetraphenylphosphonium (TPP⁺). In the absence of membrane potential, the amounts of binding were proportional to the probe concentration in the medium when the concentration is dilute. Upon illumination, interior negative membrane potential is generated which induces the uptake of phosphonium cation probe. 2 μM were employed as the initial probe concentration. The real membrane potential was evaluated by means of extrapolation to the state of no binding: The values of for various probes are plotted against the binding coefficient. Here, C_i^app is the apparent intra-vesicular concentration of the probes which is calculated without consideration of bound probes. The ordinate intercept of the plot gives the true concentration ratio, and from this the membrane potential is evaluated. The membrane potential-dependent binding was analysed with a model: the membrane is split into two halves, outer and inner half, and the amounts of bound probes in each region are governed by the concentration in the contiguous solution. We obtained a formula which describes amounts of binding as a function of the membrane potential.     

Na+-independence of the stability under alkaline conditions of the membrane of an alkalophilic Bacillus

Abstract The stability under alkaline conditions of the membrane of the alkalophile was studied. By an alkaline treatment in the absence of Na⁺ or Li⁺, the abilities of the membrane vesicles, when energized with ascorbate plus tetramethylphenylenediamine, to produce a membrane potential (negative, inside) and transmembrane pH gradient (outside > inside) were rapidly lost. The activity of cytochrome oxidase was not affected by the alkaline treatment irrespective of the presence of Na⁺. It is likely that the membrane structure is sensitive to an alkaline pH and maintained specifically by the presence of Na⁺ (or Li⁺) in the alkaline medium.     

Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp.

The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp. strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles. Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8. Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi. As the internal H+ concentration increased, the Na+ efflux reaction was inhibited. This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction. These results have also been observed in delta pH-driven Na+ efflux experiments. When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence. The results of H+ influx experiments showed a good coincidence with those of Na+ efflux. H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration. These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.     

Comparison of the energetics of lactose active transport: artificial versus enzyme-associated energy source

To further consider the thermochemical method as a useful approach for active transport research and to investigate the characteristic of a proton electrochemical potential (delta mu H+) across the membrane, the energetics of lactose active transport across Escherichia coli membrane vesicles coupled with an artificial electron donor (phenazine methosulfate-ascorbate) has been investigated. The results were compared with those obtained with an enzyme-associated electron donor (lactate dehydrogenase-D-lactate). The oxidation of an electron donor provided the energy necessary for the transport process. The observed higher heat of ascorbate oxidation reaction in the presence of a proton ionophore (carbonyl cyanide m-chlorophenylhydrazone) further confirmed the formation of delta mu H+ across the membrane. Part of the oxidation energy was utilized to form delta mu H+. Comparison of the energetics revealed that the magnitudes of delta Hox (the enthalpy of the oxidation reaction) and delta Hm (the enthalpy of the formation of delta mu H+) in the two energy sources were comparable (-46 kcal/mol of ascorbate to -40 kcal/mol of D-lactate for delta Hox and 9.6 kcal/mol of ascorbate to 14 kcal/mol of D-lactate for delta Hm). Comparable and low value (about 1%) was also found in the free energy transfer (defined by delta Gm/delta Gox) from the oxidation reaction to the formation of delta mu H+. These results, in combination with the close values of delta mu H+ observed in the two systems, suggested that the characteristic of the created delta mu H+ was independent of the energy source. Examination of delta Hm might provide the information on the ratio of the number of protons produced, as 1 mol of two different electron donors was oxidized. The oxidation reaction in the presence of membrane vesicles was discussed.     

Cytochrome o type oxidase from Escherichia coli. Characterization of the enzyme and mechanism of electrochemical proton gradient generation

Cytochrome o type oxidase purified from the membrane of Escherichia coli consists of four polypeptides (Mr 66000, 35000, 22000, and 17000), and the monomeric form predominates in octyl beta-D-glucopyranoside. The oxidase complex contains two b-type cytochromes (b-558 and b-563) and 2 mol of heme/mol of enzyme. Cytochrome o utilizes ubiquinol-1 and a number of other artificial electron donors as substrates but does not oxidize reduced cytochrome c or ferrocyanide. Activity is highly dependent upon exogenous phospholipids and/or Tween 20, and the quinone analogues 2-heptyl-4-hydroxyquinoline N-oxide and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole are potent inhibitors. Proteoliposomes were formed by detergent dilution or dialysis in the presence of the oxidase and phospholipids, followed by freeze-thaw/sonication. Vesicles formed by this means are unilamellar and contain a random distribution of 85-90-A intramembranous particles on the convex and concave fracture surfaces. During oxidase turnover, the reconstituted system generates a proton electrochemical gradient (interior negative and alkaline) of -115 to -140 mV; however, respiratory control is minimal (i.e., respiratory control ratios of about 1.5 are observed). By using a glass electrode to measure changes in external pH and the fluorescence of entrapped 8-hydroxy-1,3,6-pyrenetrisulfonate to measure changes in internal pH, it is apparent that during ubiquinol oxidation, protons are released on the external surface of the membrane and consumed on the internal surface. In contrast, with N,N,N',-N'-tetramethyl-p-phenylenediamine, an electron donor that carries few protons at neutral pH, little change in external pH is observed until the protonophore carbonyl cyanide m-chlorophenylhydrazone is added, at which point the medium becomes alkaline. The results taken as a whole are consistent with the concept that oxidase turnover generates an electrical potential (interior negative) due to vectorial electron flow from the outer to the inner surface of the membrane. The pH gradient (interior alkaline), on the other hand, appears to result from scalar (i.e., nonvectorial) reactions that consume and release protons at the inner and/or outer surfaces of the membrane, respectively. In other words, cytochrome o oxidase from Escherichia coli does not appear to catalyze vectorial proton translocation.     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

Determination of transmembrane pH gradients and membrane potentials in liposomes.

Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm.     

Relationships between the Na+-H+ antiport activity and the components of the electrochemical proton gradient in Escherichia coli membrane vesicles

The kinetics of Na+ efflux from Escherichia coli RA 11 membrane vesicles taking place along a favorable Na+ concentration gradient are strongly dependent on the generation of an electrochemical proton gradient. An energy-dependent acceleration of the Na+ efflux rate is observed at all external pHs between 5.5 and 7.5 and is prevented by uncoupling agents. The contributions of the electrical potential (delta psi) and chemical potential (delta pH) of H+ to the mechanism of Na+ efflux acceleration have been studied by determining the effects of (a) selective dissipation of delta psi and delta pH in respiring membrane vesicles with valinomycin or nigericin and (b) imposition of outwardly directed K+ diffusion gradients (imposed delta psi, interior negative) or acetate diffusion gradients (imposed delta pH, interior alkaline). The data indicate that, at pH 6.6 and 7.5, delta pH and delta psi individually and concurrently accelerate the downhill Na+ efflux rate. At pH 5.5, the Na+ efflux rate is enhanced by delta pH only when the imposed delta pH exceeds a threshold delta pH value; moreover, an imposed delta psi which per se does not enhance the Na+ efflux rate does contribute to the acceleration of Na+ efflux when imposed simultaneously with a delta pH higher than the threshold delta pH value. The results strongly suggest that the Na+-H+ antiport mechanism catalyzes the downhill Na+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Studying the drift of in line pH measurements in cell culture

The culture of Chinese hamster ovary (CHO) cells to produce monoclonal antibodies (MAb) requires accurate measurement and control of pH. Unwanted pH drifts in cell culture can adversely affect process performance, product quality, and product yield. To measure and control pH throughout the length of a culture, most cell culture processes use traditional glass pH probes. Several variables can affect the design and performance of glass pH electrodes and lead to drift in the measurement. Understanding these variables and their effects on pH performance can lead to design improvements and potentially reduce the drift. In this study, a set of Rosemount Analytical glass pH probes was investigated in cell culture operations. Electrochemical properties of the probes were monitored throughout the experiments. Experimental results show that the glass membrane potential experiences the biggest change during cell culture operations. Changes in the reference electrode potential are small compared with the changes in glass membrane potential. The glass membranes are affected by the steam sterilization process and this is the main cause for drift in the probe sensing signal during cell culture operations. Steam sterilization can cause the potential of glass membranes to change by up to 15 mV (～ 0.25 pH units). This change in membrane potential can be observed as an undesirable pH drift in bioreactors.