Influx and efflux kinetics of cationic dye binding to respiring mitochondria.

We have investigated the kinetics of interaction of cationic fluorescent lipophiles (dyes) rhodamine 123, rhodamine 6G, tetramethyl rhodamine ethyl ester, safranine O, 1,1'-diethyloxacarbocyanine, 1,1'-diethyloxadicarbocyanine, and 1,1'-diethylthiadicarbocyanine iodide with isolated respiring rat-liver mitochondria (RLM). Dye flux across the RLM inner membrane was measured by following the kinetics of fluorescence signal change after mixing of dye and RLM. The time course of fluorescence was analysed in terms of a kinetic model of the binding and transport processes involved. The rate constants of dye influx and efflux were extracted from the observed effect on the apparent time constant of fluorescence change to equilibrium intensity upon mixing dye with increasing concentrations of RLM. From the influx rate constants obtained, the apparent permeability constants for dye influx (at zero potential) across the membrane were calculated and ranged from 3 to 140 x 10(-4) cm/s. The influx rate constant was found to be linearly related to relative dye lipophilicity, as predicted by the model. As another test of the model, from the ratio of the influx and efflux rate constants, the apparent trans-membrane potential, psi, was calculated and found generally to agree with reported values, but to depend on the lipophilicity of the dye used. Not predicted by the simple model was a dissymmtry observed in the influx and efflux time constants for fluorescence change to equilibrium intensity. Inferences are made relating to the utility of these dyes as probes of psi.     

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 μM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 μM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ→0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 μg).     

Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3′-diheptyloxacarbocyanine iodide [DiOC₇(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.     

Intramitochondrial accumulation of cationic Atto520-biotin proceeds via voltage-dependent slow permeation through lipid membrane

Conjugation to penetrating cations is a general approach for intramitochondrial delivery of physiologically active compounds, supported by a high membrane potential of mitochondria having negative sign on the matrix side. By using fluorescence correlation spectroscopy, we found here that Atto520-biotin, a conjugate of a fluorescent cationic rhodamine-based dye with the membrane-impermeable vitamin biotin, accumulated in energized mitochondria in contrast to biotin-rhodamine 110. The energy-dependent uptake of Atto520-biotin by mitochondria, being slower than that of the conventional mitochondrial dye tetramethyl-rhodamine ethyl ester, was enhanced by the hydrophobic anion tetraphenylborate (TPB). Atto520-biotin also exhibited accumulation in liposomes driven by membrane potential resulting from potassium ion gradient in the presence valinomycin. The induction of electrical current across planar bilayer lipid membrane by Atto520-biotin proved the ability of the compound to permeate through lipid membrane in a cationic form. Atto520-biotin stained mitochondria in a culture of L929 cells, and the staining was enhanced in the presence of TPB. Therefore, the fluorescent Atto520 moiety can serve as a vehicle for intramitochondrial delivery of hydrophilic drugs. Of importance for biotin-streptavidin technology, binding of Atto520-biotin to streptavidin was found to cause quenching of its fluorescence similar to the case of fluorescein-4-biotin.     

The use of a cyanine dye in measuring membrane potential in yeast

An attempt was made to use 3,3'-dipropylthiacarbocyanine as a membrane potential probe in yeast by following both its fluorescence changes and its uptake by the cells under different conditions. It was found that the uptake of the dye into the cytoplasmic compartment was translated into an increased fluorescence, and the uptake by the mitochondria produced a quenching of the fluorescence. The experiments to measure uptake showed that a large amount of the dye was taken up by the cells under "deenergized" conditions. The uptake of the cyanine, however, was significantly reduced by the omission of the substrate, by deenergization of the mitochondria, or by the addition of K+, but not by Na+. This cyanine seems to be a good, qualitative indicator of the potential of the plasma membrane and of the mitochondria of the cells, with a faster response than those probes used before in yeast.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to delta pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 microM) and ATPase activity of inverted inner membrane vesicles (Ki = 126 microM) and partially purified F1-ATPase (Ki = 177 microM). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 microM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. delta psi-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of delta psi in isolated mitochondria.     

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.     

Generation of a membrane potential by sodium-dependent succinate efflux in Selenomonas ruminantium.

When Selenomonas ruminantium HD4 was grown in a chemostat, maximal succinate production and the highest molar growth yield values were both observed at a dilution rate of roughly 0.2 h-1. To determine the possible relationship between succinate efflux and high molar growth yields, the generation of a membrane potential by succinate efflux was studied in whole cells and vesicles (inside-out and right-side-out) prepared from S. ruminantium. Washed whole cells took up succinate in the absence of an exogenous energy supply; uptake was completely abolished by brief treatment with dinitrophenol or with nigericin and valinomycin. High levels of sodium ions (with respect to the intracellular sodium concentration in the assay buffer had a stimulatory effect on succinate uptake. When succinate was added to inside-out vesicles, a membrane potential (inside positive) was generated, as indicated by fluorescence quenching of the anionic lipophilic dye Oxonol V. Fluorescence quenching was sensitive to uncoupling by gramicidin D but only partially sensitive to the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In right-side-out vesicles, succinate uptake could be driven by an artificially imposed sodium gradient but not by a potassium diffusion potential; imposition of both a sodium gradient and potassium diffusion potential resulted in improved succinate uptake. The generation of a membrane potential (inside negative) upon succinate efflux was demonstrated directly in right-side-out vesicles when succinate-loaded vesicles were diluted into succinate-free buffer, and the lipophilic cationic probe tetraphenylphosphonium accumulated in the vesicles. Results indicate that an electrogenic succinate-sodium symporter is present in S. ruminantium. Transport of succinate out of the cell via the symporter might be responsible for the high molar growth yields obtained by this organism when it is grown at dilution rates where maximal succinate production occurs.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.     

Formation of a leakage-type ion pathway in lipid bilayer membranes by divalent cationic cyanine dyes in cooperation with inorganic phosphate. Role of the cyanine dye in uncoupling of oxidative phosphorylation

Divalent cationic cyanine dyes, such as 2,2'-[3-[2-(3-butyl-4-methyl-2-thiazoline-2-ylidene)-ethylidene] propenylene]bis[3-butyl-4-methylthiazolinium iodide] and platonin induced large membrane current fluctuation when an electrical potential difference was applied across a planar phosphatidylserine bilayer membrane in medium containing inorganic phosphate (Pi). Without Pi, the dyes did not induce current fluctuation at concentrations of less than 30 microM. Noise analysis of current fluctuation indicated formation of a pathway for ion leakage. From measurements of the interfacial tension between oil and aqueous phases, and of water permeability across liposomal membranes, Pi was concluded to relax the phospholipid bilayer structure, resulting in great reduction in the concentration of the cyanine dye necessary for induction of the leakage-type pathway. In the presence of Pi, cationic ions such as tetrabutyl ammonium and tetraphenyl phosphonium did not induce the leakage-type pathway, although they had electrophoretic effects at high concentrations. These facts suggest that the mechanism of the uncoupling of oxidative phosphorylation by dicationic cyanine dyes in mitochondria is different from that of cationic uncouplers such as tetrabutyl ammonium ion.     

Effects of differently hydrophobic solvents on the aggregation of cationic dyes as measured by quenching of fluorescence and/or metachromasia of the dyes

Aggregation of some cationic dyes leads to the appearance of their metachromatic spectra and/or quenching of their fluorescence. Ethanol and urea destroy metachromasia and enhance the fluorescence of such dyes by disaggregating them, suggesting hydrophobic bonds to be involved in their aggregation as in the formation of soap micelles or globular proteins. The ability of alcohols to disaggregate cationic dyes has been shown to be increased in the series methanol, ethanol, iospropanol and tertiary-butanol which is the order of increasing hydrophobic character of the alcohols themselves. Dimethyl urea is shown to be more effective than urea in destroying the metachromasia of toluidine blue, thus supporting the idea of hydrophobic bond to be involved in dye aggregation. Rhodamine 6 G undergoes quenching of its fluorescence in presence of a suitable polyanion but it is not metachromatic like acridine orange. Since only some specific cationic dyes undergo spectral changes with aggregation such changes seem to be the secondary effects of aggregation.Pool Officer, Scientists' Pool, Council of Scientific & Industrial Research (India), attached to the Bose Institute.     

Cyanine and safranine dyes as membrane potential probes in cytochrome c oxidase reconstituted proteoliposomes

Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.     

Interaction of fluorescent berberine alkyl derivatives with respiratory chain of rat liver mitochondria

The cationic fluorescent dyes, berberines, have been observed to inhibit NAD-linked respiration in rat liver mitochondria. Low concentrations inhibit electron transport in the NAD-ubiquinone span after penetration into mitochondria. More hydrophobic alkyl derivatives proved to be stronger inhibitors showing more rapid onset of inhibition. The inhibition was totally dependent on the energization of the membrane; however, the addition of a hydrophobic anion stimulated the inhibition effects in uncoupled mitochondria. Substantially higher concentrations of berberines are needed for the inhibition of the oxidation of succinate. The excess of dye interacting with surface dipoles in the energized state can inhibit the energy transduction through the complexbc ₁. On the basis of the difference in the rate of fluorescence response when berberines are added to coupled mitochondria and the corresponding inhibition effects, the presence minimally of two binding sites was suggested. The dye bound on the outer surface is highly fluorescent and inhibits the energy transduction if added in excess. The remaining dye interacting with NADH dehydrogenase does not fluoresce. The accumulation of alkylberberine in mitochondria results in additional effects in the region of cytochromeb the nature of which is not fully understood.     

The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria

The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.     

Ion channel-mediated uptake of cationic vital dyes into live cells: a potential source of error when assessing cell viability

Ionic “vital dyes” are commonly used to assess cell viability based on the idea that their permeation is contingent on a loss of membrane integrity. However, the possibility that dye entry is conducted into live cells by endogenous membrane transporters must be recognized and controlled for. Several cation-selective plasma membrane-localized ion channels, including the adenosine 5?-triphosphate (ATP)-gated P2X receptors, have been reported to conduct entry of the DNA-binding fluorescence dye, YO-PRO-1, into live cells. Extracellular ATP often becomes elevated as a result of release from dying cells, and so it is possible that activation of P2X channels on neighboring live cells could lead to exaggerated estimation of cytotoxicity. Here, we screened a number of fluorescent vital dyes for ion channel-mediated uptake in HEK293 cells expressing recombinant P2X2, P2X7, or TRPV1 channels. Our data shows that activation of all three channels caused substantial uptake and nuclear accumulation of YO-PRO-1, 4?,6-diamidino-2-phenylindole (DAPI), and Hoechst 33258 into transfected cells and did so well within the time period usually used for incubation of cells with vital dyes. In contrast, channel activation in the presence of propidium iodide and SYTOX Green caused no measurable uptake and accumulation during a 20-min exposure, suggesting that these dyes are not likely to exhibit measurable uptake through these particular ion channels during a conventional cell viability assay. Caution is encouraged when choosing and employing cationic dyes for the purpose of cell viability assessment, particularly when there is a likelihood of cells expressing ion channels permeable to large ions.     

Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and DPPAO

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.     

Mycoplasma membrane potentials determined by potential-sensitive fluorescent dyes

The membrane potentials of mycoplasmas were investigated by using potential-sensitive cyanine dyes. The fluorescence response results from a potential-dependent partition of the dyes between the cells and the extracellular medium. Cell hyperpolarization (inside more negative), e.g., by the addition of valinomycin, results in uptake of the dyes into the cells and, by formation of dye aggregates, in quenching of the fluorescence intensity. The magnitude of the fluorescence change upon addition of valinomycin depended on the external K⁺ concentration. At a defined external K⁺ concentration, no change in fluorescence occurred. The intracellular K⁺ concentration was determined by atomic absorption spectroscopy. Mycoplasma membrane potentials were calculated according to the Nernst equation. The membrane potential of bothMycoplasma mycoides subsp.capri andMycoplasma gallisepticum was −48 mV±10%; the membrane potential ofAcholeplasma laidlawii was −28 mV±20%.     

Leakage of membrane vesicle contents: determination of mechanism using fluorescence requenching.

Agents such as antimicrobial peptides and toxins can permeabilize membrane vesicles to cause leakage of entrapped contents in either a graded or an all-or-none fashion. Determination of which mode of leakage is induced is an important step in understanding the molecular mechanism of membrane permeabilization. Wimley et al. (1994, Protein Sci. 3:1362-1378) have developed a fluorescence method for distinguishing the two modes that makes use of the dye/quencher pair 8-aminonapthalene-1,3,6 trisulfonic acid (ANTS)/p-xylene-bis-pyridinium bromide (DPX) without the usual need for the physical separation of vesicles from released contents. Their "requenching" method establishes the mode of release through the fluorescence changes that occur when DPX is added externally to a solution of vesicles that have released some fraction of their contents. However, the requenching method as originally stated ignored the possibility of preferential release of dye or quencher. Here we extend the theory of the method to take into account preferential release and the effects of graded leakage. The ratio of the rates of release of the cationic quencher DPX and anionic dye 8-aminonapthalene-1,3,6 trisulfonic acid can be estimated by means of the theory. For graded leakage, we show that the release of the markers does not coincide with the fluorescence changes observed in the standard leakage assay. This is true for self-quenching dyes as well and means that 1) the amount of released material will be overestimated and 2) the kinetics will be nonexponential and have artificially high apparent rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy transfer in lipid bilayers.

The quenching of fluorescence due to energy transfer between a dilute, random array of donor and acceptor chromophores in lipid bilayer was measured and compared to theoretical expressions developed to predict the decrease in emission intensity under these circumstances. The observed intensity was found to be the same function of quencher concentration in both planar, multilamellar dispersions and small, spherical vesicles. The degree of quenching was accurately predicted by a simple relation derived in this paper, as well as a more complex equation previously developed by Tweet, et al. The results suggest that significant quenching may be observed even when the average donor-acceptor separation exceeds the F?rster critical distance by severalfold. Application of these results to problems of current interest in membrane research are discussed.     

Plant mitochondrial electrical potential monitored by fluorescence quenching of rhodamine 123

The suitability of the fluorescent dye rhodamine 123 for qualitative and quantitative determinations of the electrical potential difference (ΔΨ) in isolated pea (Pisum sativum L.) stem mitochondria was evaluated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both external and internal substrates. This quenching was associated to the generation of ΔΨ, because it was completely released by uncouplers and respiratory inhibitors. The conversion of the proton gradient (ΔpH) into ΔΨ, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was accompanied by 40 % of unspecific binding in coupled, but not in uncoupled, mitochondria. Rhodamine 123 quenching varied linearly with a K+-diffusion potential. ADP induced a transient and cyclic change of fluorescence which was associated to ATP synthesis. Consequently, rhodamine 123 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the probe was not toxic. It is concluded that rhodamine 123, followed by fluorescence quenching, is a suitable probe to study the energetics of isolated plant mitochondria. This revised version was published online in July 2006 with corrections to the Cover Date.