细胞内pH值测定方法的研究进展

很多细胞的活动都对pH的变化十分敏感。pH值的有效控制对维持正常的细胞生理活动十分重要。如何有效监测细胞内pH值是很多细胞生物学的重要研究内容之一,如在研究细胞内转运蛋白、Ca2+离子等的变化活动时均需要测定细胞内的pH值,其相关的研究已有100多年的历史。本文将介绍目前几种细胞内pH值的主要测定方法,包括:弱酸弱碱分布法、核磁共振法、微电极法、荧光探针法等;每一种方法将从相关方法、技术的原理、特点、应用、局限性和注意事项等着手,将各个方法的优缺点进行横向的比较。本文还将重点探讨免疫探针法的最新进展,将报道一种最新的基于碳纳米点技术和荧光染料结合的pH定量测定计,还将介绍SNARF的两种最新衍生染料SNARF-F和SNARF-Cl的性能及其应用。     

Calibration of the response of 9-amino acridine fluorescence to transmembrane pH differences in bacterial chromatophores

The spectral characteristics of absorption and fluorescence emission of 9-amino acridine are not altered by the interaction with bacterial chromatophores, except for the attenuation of both the absorption and emission following the formation of a protonic gradient. The lifetime of fluorescence of the dye is significantly affected in the presence of membranes, and even more following illumination. The shortening of the lifetime induced by light is reversible and prevented by nigericin and K+. The onset kinetics of the fluorescence quenching following the generation of an artificial transmembrane pH difference is temperature dependent, with an activation energy of 17 +/- 3 kcal/mol. The effect of pH on the rate constants is consistent with a model assuming that the diffusion of the unprotonated species is the limiting step in the quenching phenomenon. The response of 9-amino acridine to artificially imposed delta pH's has been utilized as a calibration method for the measurements of the light-induced protonic gradient. The apparent inner volume of chromatophores, evaluated from the extraplation of the response at delta pH = 0, was found to be much larger (15- to 40-fold) than the true osmotic volume, indicating that most of the dye is bound to the membrane when accumulated into the inner lumen.     

Properties of a pigment system consisting of carotenoids and reaction centre pigments in chromatophores of Rhodospirillum rubrum

A pigment system containing carotenoids and oxidised reaction centre pigments is present in chromatophores of Rhodospirillum rubrum and this pigment system may cause fluorescence quenching when a still unidentified chromatophore component is in its oxidised state. Besides by its action spectrum, this pigment system is characterised by the time course and level of light saturation of the effect. The quenching of bacteriochlorophyll fluorescence is abolished when the permeability of the chromatophore membranes is affected. The quenching effect is correlated with a reversible absorption decrease of B 880. A possible function for this pigment system is discussed.     

Adaptation of soil bacterial communities to prevailing pH in different soils

Abstract: The bacterial community response to pH was studied for 16 soils with pH(H₂O) ranging between 4 and 8 by measuring thymidine incorporation into bacteria extracted from the soil into a solution using homogenization-centrifugation. The pH of the bacterial solution was altered to six different values with dilute sulfuric acid or different buffers before measuring incorporation. The resulting pH response curve for thymidine incorporation was used to compare bacterial communities from the different soils. There was a correlation between optimum pH for thymidine incorporation and the soil pH(H₂O). Even bacterial communities from acid soils had optima corresponding to the soil pH, indicating that they were adapted to these conditions. Thymidine incorporation was also compared with leucine incorporation for some soils. The leucine to thymidine incorporation ratio was constant over the tested pH interval when incorporation values were adjusted for isotope dilution. A good correlation was found between the scores along the first component (explaining 80% of the variation) and soil pH ( r ² = 0.85), if principal component analysis of the pH response curves for thymidine incorporation was used. The pH response curves differed most for the extreme pH values used, and a linear relationship was found between the logarithm of the ratio of thymidine incorporation at pH 4.3 to incorporation at pH 8.2 and the soil pH ( r ² = 0.86). Thus, a simplified technique using only two pH values, when measuring the thymidine incorporation, could be used to compare the response to pH of bacterial communities.     

Anacardic acid-mediated changes in membrane potential and pH gradient across liposomal membranes

We have previously shown that anacardic acid has an uncoupling effect on oxidative phosphorylation in rat liver mitochondria using succinate as a substrate (Life Sci. 66 (2000) 229-234). In the present study, for clarification of the physicochemical characteristics of anacardic acid, we used a cyanine dye (DiS-C3(5)) and 9-aminoacridine (9-AA) to determine changes of membrane potential (ΔΨ) and pH difference (ΔpH), respectively, in a liposome suspension in response to the addition of anacardic acid to the suspension. The anacardic acid quenched DiS-C3(5) fluorescence at concentrations higher than 300 nM, with the degree of quenching being dependent on the log concentration of the acid. Furthermore, the K⁺ diffusion potential generated by the addition of valinomycin to the suspension decreased for each increase in anacardic acid concentration used over 300 nM, but the sum of the anacardic acid- and valinomycin-mediated quenching was additively increasing. This indicates that the anacardic acid-mediated quenching was not due simply to increments in the K⁺ permeability of the membrane. Addition of anacardic acid in the micromolar range to the liposomes with ΔΨ formed by valinomycin-K⁺ did not significantly alter 9-AA fluorescence, but unexpectedly dissipated ΔΨ. The ΔΨ preformed by valinomycin-K⁺ decreased gradually following the addition of increasing concentrations of anacardic acid. The ΔΨ dissipation rate was dependent on the pre-existing magnitude of ΔΨ, and was correlated with the logarithmic concentration of anacardic acid. Furthermore, the initial rate of ΔpH dissipation increased with logarithmic increases in anacardic acid concentration. These results provide the evidence for a unique function of anacardic acid, dissimilar to carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin, in that anacardic acid behaves as both an electrogenic (negative) charge carrier driven by ΔΨ, and a ‘proton carrier’ that dissipates the transmembrane proton gradient formed.     

Macroscopic optical mapping of excitation in cardiac cell networks with ultra-high spatiotemporal resolution

Optical mapping of cardiac excitation using voltage- and calcium-sensitive dyes has allowed a unique view into excitation wave dynamics, and facilitated scientific discovery in the cardiovascular field. At the same time, the structural complexity of the native heart has prompted the design of simplified experimental models of cardiac tissue using cultured cell networks. Such reduced experimental models form a natural bridge between single cells and tissue/organ level experimental systems to validate and advance theoretical concepts of cardiac propagation and arrhythmias. Macroscopic mapping (over >1cm(2) areas) of transmembrane potentials and intracellular calcium in these cultured cardiomyocyte networks is a relatively new development and lags behind whole heart imaging due to technical challenges. In this paper, we review the state-of-the-art technology in the field, examine specific aspects of such measurements and outline a rational system design approach. Particular attention is given to recent developments of sensitive detectors allowing mapping with ultra-high spatiotemporal resolution (>5 megapixels/s). Their interfacing with computer platforms to match the high data throughput, unique for this new generation of detectors, is discussed here. This critical review is intended to guide basic science researchers in assembling optical mapping systems for optimized macroscopic imaging with high resolution in a cultured cell setting. The tools and analysis are not limited to cardiac preparations, but are applicable for dynamic fluorescence imaging in networks of any excitable media.     

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.     

Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 10⁸ cells (g soil, dry wt.)^–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 10⁸ cells (g soil, dry wt.)^–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 10⁸ cells (g soil, dry wt.)^–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments. Received: 29 March 1997 / Accepted: 28 May 1997     

Selective labeling and monitoring pH changes of lysosomes in living cells with fluorogenic pH sensors

New BODIPY-based pH probes have been designed with excitation and emission wavelengths suitable for fluorescence microscopy and flow cytometry. These pH probes are cell-permeable, selectively label lysosomes, and can be used for noninvasive monitoring of lysosomal pH changes during physiological and pathological processes.     

Influence of pH value and salts on the adsorption of lysozyme in mixed‐mode chromatography

Mixed‐mode chromatography (MMC) is an interesting technique for challenging protein separation processes which typically combines adsorption mechanisms of ion exchange (IEC) and hydrophobic interaction chromatography (HIC). Adsorption equilibria in MMC depend on multiple parameters but systematic studies on their influence are scarce. In the present work, the influence of the pH value and ionic strengths up to 3000 mM of four technically relevant salts (sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate) on the lysozyme adsorption on the mixed‐mode resin Toyopearl MX‐Trp‐650M was studied systematically at 25℃. Equilibrium adsorption isotherms at pH 5.0 and 6.0 were measured and compared to experimental data at pH 7.0 from previous work. For all pH values, an exponential decay of the lysozyme loading with increasing ionic strength was observed. The influence of the pH value was found to depend significantly on the ionic strength with the strongest influence at low ionic strengths where increasing pH values lead to decreasing lysozyme loadings. Furthermore, a mathematical model that describes the influence of salts and the pH value on the adsorption of lysozyme in MMC is presented. The model enables predicting adsorption isotherms of lysozyme on Toyopearl MX‐Trp‐650M for a broad range of technically relevant conditions.     

On the functional unit of energy coupling in photophosphorylation by bacterial chromatophores

(1) Putative relationships between the rate of photophosphorylation, the proton-motive force and the concentration of an uncoupling molecule are considered within the framework of the delocalised chemiosmotic coupling hypothesis. The addition of a partially inhibitory titre of a specific, tight-binding H⁺-ATP synthase inhibitor is not expected, within the framework of a delocalised coupling model, to alter the form of this relationship. (2) Photophosphorylation in chromatophores from Rhodopseudomonas capsulata is potently uncoupled by the protonophore SF6847. Yet the uncoupling potency of this compound is actually further increased when the rate of phosphorylation in the absence of protonophore is decreased by the addition of the energy-transfer inhibitor venturicidin, in contrast to the expectations of a delocalised energy-coupling model. (3) Similarly, valinomycin (in the presence of nigericin) uncouples more potently when the number of active H⁺-ATP synthases is decreased by the addition of the energy -transfer inhibitors N,N′-dicyclohexyl-carbodiimide or venturicidin. (4) The pore-forming ionophore gramicidin D also uncouples photophosphorylation more potently when the number of active H⁺-ATP synthases is reduced. (5) These results are discussed in relation to the idea that the functional unit of electrical events and photophosphorylation either is, or is not, the intact membrane vesicle. (6) It is concluded that the unit of energy coupling in bacterial chromatophores is much smaller than the entire coupling membrane vesicle, and that previous analyses of this point, based on titrations with ionophores alone, may need to be re-examined.     

Photoelectric effects in bacterial chromatophores. Comparison of spectral and direct electrometric methods

Generation of photoelectric potential in chromatophores of Rhodopseudomonas sphaeroides has been measured (i) spectrophotometrically, using electrochromic shift of carotenoid absorption band or (ii) electrometrically, by means of two electrodes separated by a collodion film covered on one side with chromatophores. A 15 ns laser flash was used to induce a single turnover of photosynthetic reaction centers. It was found that results obtained by both methods are similar in (i) direction of electric vector (the chromatophore interior positive) and (ii) redox titration curves (E_m = 10mV). The magnitudes of the photopotential were about 60 and 25 mV, when monitored with spectral and electrometric techniques, respectively. In both cases, the rise times of the photopotentials were faster than time resolution of the techniques used. Decay of the response of carotenoids was found to be slower than that in the collodion film system. The addition of ubiquinone Q₁₀ into the decane solution of asolectin used to impregnate the collodion film led to slowing down of the decay. The carotenoid response decay could be accelerated by FCCP or o-phenanthroline. In the latter case, the shape of the decay curve coincides with decay of the photopotential measured in the collodion film system. It is suggested that decane extracts secondary ubiquinone from chromatophores attached to the collodion film. Such an unfavorable effect can be strongly decreased by added ubiquinone     

Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae

Abstract Fluorescence ratio imaging microscopy using pH-sensitive fluorescent dyes makes it possible to evaluate statistical distribution of intracellular pH in a population of the yeast S. cerevisiae examined in a thin layer of suspension in a Petri dish. The distribution appears to fit a Gaussian curve with a half-width around the 0.4 pH unit. The curve became slightly narrower after resuspension in a strong buffer; the mean values shifted with the pH of the buffer. The shape of the distribution curves of both resting and growing cells in various phases of growth does not change significantly. Likewise, addition of 1% of glucose, 50 μM suloctidil or 100 μM diethylstilbestrol brings about no alteration. The only value which clearly changes is the average cytoplasmic pH.     

Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis

ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.     

The redox midpoint potential of the primary quinone of reaction centers in chromatophores of Rhodobacter sphaeroides is pH independent

The redox midpoint potential (E (m)) of the primary quinone of bacterial reaction centers, Q(A), in native membranes (chromatophores) measured by redox potentiometry is reported to be pH dependent (-60 mV/pH) up to a highly distinctive pK ( a ) (9.8 in Rba. sphaeroides) for the reduced state. In contrast, the E (m) of Q(A) in isolated RCs of Rba. sphaeroides, although more variable, has been found to be essentially pH-independent by both redox potentiometry and by delayed fluorescence, which determines the free energy (DeltaG (P*A)) of the P(+)Q (A) (-) state relative to P*. Delayed fluorescence was used here to determine the free energy of P(+)Q (A) (-) in chromatophores. The emission intensity in chromatophores is two orders of magnitude greater than from isolated RCs largely due to the entropic effect of antenna pigments "drawing out" the excitation from the RC. The pH dependence of DeltaG (P*A) was almost identical to that of isolated RCs, in stark contrast with potentiometric redox titrations of Q(A). We considered that Q(A) might be reduced by disproportionation with QH(2) through the Q(B) site, so the titration actually reflects the quinone pool, giving the -60 mV/pH unit dependence expected for the Q/QH(2) couple. However, the parameters necessary to achieve a strong pH-dependence are not in good agreement with expected properties of Q(A) and Q(B). We also consider the possibility that the time scale of potentiometric titrations allows the reduced state (Q (A) (-) ) to relax to a different conformation that is accompanied by stoichiometric H(+) binding. Finally, we discuss the choice of parameters necessary for determining the free energy level of P(+)Q (A) (-) from delayed fluorescence emission from chromatophores of Rba. sphaeroides.     

Role of external calcium in homeostasis of intracellular pH in the cyanobacterium Anabaena sp. strain PCC7120 exposed to low pH

The role of external Ca²⁺ in the homeostasis of intracellular pH (pHi) of Anabaena sp. strain PCC7120 in response to a decrease in the external pH (pHex) has been studied in cell suspensions. Increase in cytoplasmic pH after acid shock is dependent on the presence of Ca²⁺ in the medium. The observed Ca²⁺-mediated alkalization of the cytoplasm depends on the extent of the shift in external pH. Acid pH shifts resulted in an increased permeability of the cytoplasmic membrane to protons, which could be reversed by increasing the concentration of Ca²⁺ in the medium. Thus, the ability of Ca²⁺ to increase cytoplasmic pH might be correlated with an inhibition of net proton uptake by increasing concentrations of external Ca²⁺ under these conditions. This combined response resulted in the generation and maintenance of a larger pH gradient (ΔpH) at acid external pH values. All Ca²⁺ channel blockers tested, such as verapamil and LaCl₃, inhibited the observed Ca²⁺-mediated response. On the other hand, the Ca ionophore calcimycin (compound A23187) was agonistic, and stimulated both cytoplasmic alkalization and inhibition of net proton uptake. The protonophorous uncoupler carbonylcyanide m -chlorophenyl hydrazone, inhibited this Ca²⁺-mediated response, whereas monensin, an inhibitor of the Na⁺/H⁺ antiporter, had no significant effect. The results of the present study suggest that an influx of Ca²⁺ from the extracellular space is required for the regulation of cytoplasmic pH in Anabaena sp. strain PCC7120 exposed to low external pH values.     

Relationship of the Donnan potential to the transmembrane pH gradient in tracheal apical membrane vesicles

Summary Experiments were performed to determine the factors which contribute to the transmembrane pH gradient (pH) and the potential gradient () in apical plasma membrane vesicles isolated from bovine tracheal epithelium. As indicated by the accumulation of¹⁴C-methylamine, the vesicles maintained a pH (inside acidic) which was dependent upon the external pH. The pH was also proportional to the ionic strength of the suspending medium, suggesting that the H⁺ distribution was dictated by a Donnan potential. Measurements of the distribution of⁸⁶Rb⁺ demonstrated an electrical potential gradient across the vesicle membrane, inside negative which was proportional to the medium ionic strength. pH changed in parallel with in response to a variety of imposed conditions. These results are compatible with the existence of a H⁺ conductance in the vesicle membrane. Thus the endogenous electrical and proton gradients may be manipulated and used as a general experimental tool to complement kinetic analysis in investigations of transport mechanism using isolated vesicle preparations.     

Measurements of intra-and extracellular pH in the liverwort Conocephalum conicum during action potentials

During action potentials triggered by electricity and light, measurements of intra- and extracellular pH in the liverwort Conocephalum conicum L. were carried out by the use of antimony-filled H⁺-sensitive microelectrodes. Intracellular pH increased transiently by about 0.05 unit in the course of an action potential, while extracellular pH, measured at the surface of the thallus, remained unchanged. Switching the light off caused a transient increase in intracellular pH by less than 0.1 unit. Turning the light on produced a fast pH decrease by about 0.15 unit followed by a slow increase. When the light was intensive enough, the action potential thus triggered caused a slight increase in intracellular pH superimposed on the phase of a slow pH increment.
The magnitude and time course of the intracellular pH changes seem insufficient for a role as messenger between action potential and the up to 100% increase in the rate of respiration that has been registered in Conocephalum conicum as a consequence of excitation.