pH measurements in ultranarrow immobilized pH gradients

It is possible to measure pH values in immobilized pH gradients (IPG) when the polyacrylamide matrix is made to contain an additional, carrier ampholyte-generated pH gradient. After an IPG run, 5 mm gel segments, along the separation axis, are cut and eluted in 300 microliter of 10 mM KCl and the pH read with a standard pH meter. When using ultranarrow pH gradients, larger gel segments (ca. 265 microliter) are eluted in 900 microliter of 100 mM KCl and the pH assessed with a differential pH meter. In the latter case, either internal or external standards are used as a reference, or starting point, to convert delta pH values into an actual pH curve. The reproducibility of the system is better than +/- 0.05 pH units, with a ca. 15% error over a 0.3 pH unit span. In ultranarrow pH gradients, it is imperative to use mixtures of all commercially available carrier ampholytes, so as to smoothen conductivity and buffering capacity gaps. By the present method, it is also possible to convert a wide (2-3 pH unit) carrier ampholyte interval into a narrow (0.2-0.3 pH unit) one.     

Intracellular pH distribution and transmembrane pH profile of yeast cells

The pH-dependent fluorescence excitation of fluorescein located intracellularly and in the vicinity of cells of the yeast Saccharomyces cerevisiae and Endomyces magnusii was used to obtain local pH values at a linear resolution 0.2 micron. Cells suspended in water or in a diluted (5 mM) acidic buffer had a relatively alkaline interior (about 7.0-7.5) with pH decreasing gradually toward the periphery and further out through the cell wall to the value of the bulk solution. In slightly alkaline weak buffers the cells also showed an alkaline center and a slightly acidic ring-shaped area, but the peripheral region close to the membrane was again alkaline with pH increasing toward the bulk solution. The heterogeneity of intracellular pH was reduced or nearly abolished in starved or antimycin-treated cell. Suspension of cells in strong (200 mM) buffer resulted within 15-20 min in a nearly homogeneous pH pattern throughout the cell, attaining pH values of 5.5-7.5, depending on the pH of the buffer. Addition of glucose with concomitant pH decrease of the extracellular medium did not change appreciably the intracellular pattern for 20-30 min, except with diethylstilbestrol (inhibitor of proton-extruding ATPase) when the cell became more acidic. It appears that the delta pH measurements between the cell as a whole and the bulk solution (as are used for the calculation of the electrochemical potential of protons in proton-driven transports) are not substantiated, the probable pH difference across the plasma membrane being substantially smaller than previously supposed.     

Sensing pH?

Abstract. pH is an all-pervasive variable in the environment of phototrophs. Phototrophs as a whole, and even the single genus Dunaliella , can grow over essentially the whole range of pH values found in nature. Such a large range of pH values, combined with other chemical variations in the environment, impose a range of constraints on plant behaviour related to intracellular pH regulation, nutrient acquisition, and avoidance of toxic effects. No single genotype can grow well over the whole pH range compatible with growth of phototrophs as a whole, although some deliberately alter surface pH so as to create a 5–6 unit pH gradient over the surface related to nutrient acquisition and avoidance of toxic influences. The regulation of these various processes does not, on current evidence, involve pH-sensing by any extracellular sensor which is not part of the catalytic or regulatory mechanism of a membrane protein such as a porter.     

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) or [¹⁴C]benzoic acid ([¹⁴C]BA) between the cells and the surrounding medium. [¹⁴C]DMO was used at external pH of 5.0 to 7.5 while [¹⁴C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.     

Effect of changing venoarterial pH difference on in vivo arterial pH

Plasma pH has been postulated to change slowly in blood leaving the pulmonary capillaries because of the uncatalyzed dehydration of CO2. If so, there could be a difference between in vivo and in vitro arterial pH, the magnitude of which would be dependent on the venoarterial pH difference (v-aDpH). We tested this hypothesis in anesthetized dogs by changing v-aDpH by airway CO2 loading and by comparing arterial pH measured in vivo by a rapidly responding intravascular pH electrode with that measured in vitro by a conventional glass electrode. Using a multiple regression analysis, we found a small but significant contribution of venous pH to in vivo arterial pH, with a regression coefficient of 0.0718 (P less than 0.0001), suggesting a postcapillary increase of in vivo arterial pH. When carbonic anhydrase was inhibited by the administration of acetazolamide, the effect of venous pH on arterial pH was abolished, and a unique relationship between in vivo and in vitro arterial pH was established (regression coefficient 1.02; P greater than 0.05, comparison with unity). These results could be accounted for in a computer simulation of gas exchange among alveolus, plasma, and erythrocyte. We conclude that there exists a small but measurable postcapillary increase in arterial pH.     

Quantitative pH assessment of small-volume samples using a universal pH indicator

We developed a hue-based pH determination method to analyze digital images of samples in a 384-well plate after the addition of a universal pH indicator. The standard error of calibration for 69 pH standards was 0.078 pH units, and no sample gave an error greater than 0.23 units. We then used in-solution isoelectric focusing to determine the isoelectric point of Wnt3A protein in conditioned medium and after purification and applied the described method to assess the pH of these small-volume samples. End users may access our standard to assay the pH of their own samples with no additional calibration.     

Isoelectric focusing in immobilized pH gradients: Generation of extended pH intervals

A new technique for generatiing extended pH gradients (3–4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the ‘Varigard’-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857–862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4–8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in thepH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (β) and ionic strength (I) courses.     

Measurements of the Cytoplasmic pH of Chara corallina using Double-barrelled pH Micro-electrodes

A range of polymeric compounds was examined for their suitabilityas pressure-stabilizing agents in liquid membrane pH micro-electrodesfor intracellular use in plant cells. Of the compounds tested,mixtures of liquid proton sensor and nitrocellulose were foundto be superior to epoxy resins, polyvinylchloride and ethylcellulose. The electrical resistance of silicone rubber mixtureswas too high for micro-electrodes with tip diameters of 1.0µm. Double-barrelled micro-electrodes containing nitrocellulosemaintained excellent pH sensitivity for up to 1.0 impalementsof charophyte cells. Measurements of cytoplasmic pH were madein both internodal and whorl cells of Chora corallina over arange of experimental conditions. The response of cytoplasmicpH to rapid changes in external pH or illumination occurredover several minutes. The advantages of the use of double-barrelledpH micro-electrodes over other methods of intracellular pH measurementsuch as the distribution of weak acids (DMO), ³¹P-NMR and single-barrelledmicro-electrodes is discussed. Key words: pH micro-electrodes, cytoplasmic pH, charophytes     

Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria.

Bacteria migrate away from an acid pH and from a number of chemicals, including organic acids such as acetate; the basis for detection of these environmental cues has not been demonstrated. Membrane-permeant weak acids caused prolonged tumbling when added to Salmonella sp. or Escherichia coli cells at pH 5.5. Tethered Salmonella cells went from a prestimulus behavior of 14% clockwise rotation to 80% clockwise rotation when 40 mM acetate was added and remained this way for more than 30 min. A low external pH in the absence of weak acid did not markedly affect steady-state tumbling frequency. Among the weak acids tested, the rank for acidity (salicylate greater than benzoate greater than acetate greater than 5,5-dimethyl-2,4-oxazolidinedione) was the same as the rank for the ability to collapse the transmembrane pH gradient and to cause tumbling. At pH 7.0, the tumbling responses caused by the weak acids were much briefer. Indole, a non-weak-acid repellent, did not cause prolonged tumbling at low pH. Two chemotaxis mutants (a Salmonella mutant defective in the chemotaxis methylesterase and an E. coli mutant defective in the methyl-accepting protein in MCP I) showed inverse responses of enhanced counterclockwise rotation in the first 1 min after acetate addition. The latter mutant had been found previously to be defective in the sensing of gradients of extracellular pH and (at neutral pH) of acetate. We conclude (i) that taxes away from acid pH and membrane-permeant weak acids are both mediated by a pH-sensitive component located either in the cytoplasm or on the cytoplasmic side of the membrane, rather than by an external receptor (as in the case of the attractants), and (ii) that both of these taxes involve components of the chemotaxis methylation system, at least in the early phase of the response.