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Safranine as membrane potential probe in rat liver mitochondria
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 255-265, 1980
Safranine
as Membrane Potential Rat Liver Mitochondria
Probe in
ADRIAN0 ZANOTTI AND GIOVANNI FELICE AZZONE With the technical assistance of Paolo Veronese C.N.R.
lJnit,for
the Study of Physiology sf Mitochondria and Institzlte University qf Padua, Italy Received October 18, 1979; revised December
qfGexera1
Pathology,
11, 1979
The absorbance changes accompanying safranine uptake due either to respiration or to K+ diffusion potential are maximal at a point of equivalence between number ofdye molecules and binding sites (“saturation” point) and decrease both below and above this point. This behavior is in accord with a model where safranine uptake is followed by stacking on adjacent membrane sites below saturation and destacking on nonadjacent sites above saturation. The values of the endpoint titrations are similar for the respiration- and K’ diffusion potentialinduced safranine uptakes only at protein concentrations above saturation. Calibration of the safranine response with K+ diffusion potentials indicates a linear region at low AI/J and a deviation from linearity at high &/I. The region of linear safranine response depends on the dye/protein ratio. Increase of dye/protein ratio leads to expansion of the linear range. However calibration at high dye/protein ratios is limited by the loss of matrix K+. At constant dye/protein ratios increase of dye concentration shifts the calibration curve at lower & values. The half-time of the safranine response increases hyperbolically with the decrease of protein at constant dye concentration and with the decrease of dye concentration at constant dye/protein ratios. Uptake and binding of safranine result in mitochondrial damage with respect to degree of energy coupling, rate of electron transport, and ADP-stimulated respiration. Oxygen pulses to anaerobic mitochondria result in an electrical field which may be followed with the safranine response. The minimal amount of oxygen capable of inducing a maximal rate of safranine uptake is 0.5 natom x mg protein’. Since the rate of safranine uptake is linear with A+, this amount of oxygen corresponds to the extent of charge separation required for a steady state A$.
electrode impalement technique, optical probes are the natural candidates. Azzi et al. (3) and Jasaitis et al. (4) have proposed the use of ANS.’ The extensive disagreement in interpreting the fluorescent response of ANS has however considerably limited the application of this probe (5, 6). Cyanine dyes have been used by Laris (7) and by Kinally et aZ. (8, 9). A coherent picture on the behavior of these probes seems to emerge from the studies of Laris (cf. however Ref. (8)). Akerman and WikStrom (10) have reported a linear response
It is now generally accepted that in steady state a large H+ electrochemical gradient exists across the membrane of energy-transducing organelles and that this H+ electrochemical gradient (AWL.,) drives various energy-requiring reactions. Since the major component of A& is the membrane potential (A$) a number of techniques have been developed for the determination of A+. The most widely used procedures are based on the distribution of permeant ions (1,2) and they are thought to provide reliable indications of A$ under steady state conditions. The inadequacy of the radioisotope distribution method for the case of short transients has however prompted interest toward alternative approaches. Because of the problems arising from the use of the
’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)LpiperazineethanesuIfonic acid; EGTA, ethylene glycol bis(@aminoethyl ether)N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; prot, protein; ANS, 1 anilinonaphtalene-8sulphonate; FCCP, carbonylcyanide-ptrifluoromethoxyphenylhydrazone. 255
0003-9861/80/050255-11$02.00/0 Copyright G 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AND AZZONE
of safranine, with respect to AI/I, due to K+ and H+ diffusion. This dye had been previously shown to undergo accumulation and stacking in mitochondria during respiration (11) and in liposomes during K+ efflux (12). Safranine is a “redistribution signal” probe (13) since its spectroscopic response is due to its redistribution at the two sides of the membrane under the effect of A$. In order to test the validity of safranine as A$ probe we have investigated the linearity of the safranine response as a function of A$, and the sensitivity and time course of the response. Furthermore since the probe is a lipophilic molecule its damaging effect on the membrane has been tested. Once ascertained as to the advantages and limitations of safranine as A$ probe, we have used safranine to investigate the relationship between charge separation and rise of A$. A preliminary report of the present study has already been presented (14). EXPERIMENTAL Rat liver mitochondria were prepared as described previously (15). The last washing was carried out in an EDTA-free medium. The standard incubation medium contained 0.2 M sucrose, 20 mM Hepes pH 7.2, 2 mM phosphate-Tris, 0.1 mM EGTA-Tris, 4 PM rotenone. Respiration was initiated by the addition of 5 mM succinate. Absorbancy changes were recorded with a dual-wavelength spectrophotometer (Aminco DW2 a) at the wavelengths 520-554 nm. The net absorbance changes of safranine were calculated by taking as a reference the absorbance of a mitochondrial suspension containing the same amount of protein and the same dye concentration and supplemented with 2 pM FCCP, 80 pmol valinomycin x mgproteinl, and 50 mM KCl. These experimental conditions were selected in order to minimize the Donnan potential in anaerobicuncoupled mitochondria (10, 16). The rate of oxygen uptake was determined with a Clark electrode in a thermostated cuvette. The dimension of A+ was measured with PH]triphenylmethylphosphonium in the following manner (2, 17, 18). The mitochondrial suspension was supplemented with the radioisotopes and centrifuged after 60 s in the S12 rotor of a Sorvall RC2B superspeed centrifuge. The mitochondrial pellet was dissolved at 40°C in 10% SDS, 0.01 M NaOH, 1% mercaptoethanol, and then added to the Packard emulsifier scintillator special MI-96. The distribution of the radioactivity between pellet and supernatant was then determined in a Packard Tri-Carb 2455 liquid scintillation spectrometer. The concentration of triphenylmethylphosphonium
in the matrix was calculated by assuming a volume of 0.5 pl/mg prot-I. Rate and extents of K+ efflux during safranine equilibration were determined with the KS electrode in order to calculate exactly the K+ diffusion potential on the K+ distribution. In the presence of 100 pM [K+], the rates of K’ efflux were 13.6, 21.8, 25.4, and 30.4 nmol K+ x mg prott’ x min-I, and the extent of K+ efflux 18.0, 21.8, 24.4, and 27.5 nmol x mg prott’ at dye/protein ratios of 5, 10,20, and 40, respectively. These data support the view that, except at very high dye/protein ratios and very high values of A$, use of the values of [K’], and [K’], at the moment of the addition of valinomycin introduces a negligible error in the calculation of A$. This further follows from two considerations. First, although the initial [K+], is generally assumed as 100 mM (19), the total extent of K+ efflux in the presence of valinomycin + uncouplers is only 30 nmol X mg pro?. However K+ efflux is accompanied by osmotic shrinkage. This reduces the matrix volume and maintains [K+] relatively constant as indicated by the linearity of the rate of K+ diffusion-driven Ca2+ uptake for an extended period (19a). Second, above 300 FM [K+],, K+ efflux increases [K+], only of a few micromolar, as compared to the initial values, with a drop in AJ, of less than 4 mV. As will be shown in Fig. 5, the A+ calculated on the K+ distribution was compared with that calculated on the distribution of triphenylmethylphosphonium. In the oxygen pulse experiments the medium was bubbled with pure nitrogen. The cuvette was then covered with paraffin oil to restrict oxygen diffusion. Residual traces of oxygen were removed by letting the mitochondria stay in the parafflin oil-covered cuvette for 10 min before oxygen addition. The oxygen diffusion through the paraffin layer, from the air to the oxygendepleted medium, was measured with oxygen electrode and found to be lower than 2.5 pM 0, x min+. Mitochondrial protein was determined with the biuret using bovine serum albumin as a standard. The term dye/protein ratio in the text refers always to nanomoles of dye x milligrams of protein-‘. Safranine was a product of BDH Chemicals Ltd., purified according to Pal and Schubert (20). Aliquots were taken from a standard solution at a concentration of 5 mM. rH]Triphenylmethylphosphonium was a kind gift of Dr. R. Kabach. All other products were of the maximal purity commercially available. RESULTS
The Interaction
Mitochondrial
of Safranine Membrane
with the
The model analyzed in Fig. 1 stems from the evidence that the spectroscopic response of “redistribution signal” probes in general, and of safranine in particular, is correlated
SAFRANINE
AS MEMBRANE
+ + + + + + + +
POTENTIAL
PROBE
257
+ + + + + F + + + A
FIG. 1. Uptake and interaction of safranine with the mitochondrial membrane. “S,, free safranine as monomer; “Sv, free safranine as tlimer or higher multimers; ‘IS,,. bound safranine as monomer; ‘S,, bound safranine in stacked form. The line AB represents the line along which zT-electrons may be shared.
with aggregation or “stacking” phenomena (cf. Refs. (11, 13, 21)) after accumulation of the dye. Safranine moves from the outer to the inner space under the influence of A$ and distributes at electrochemical equilibrium, external >‘S, + matrix zlSr. The concentration of free safranine in the matrix reaches, in presence of a A$ of 120- 180mV, negative inside, the millimolar range. Part of safranine undergoes aggregation in solution, i.e., formation of dimers or of higher multimers. Part of safranine undergoes binding to membrane anionic sites. These anionic sites appear to be located in a hydrophobic environment (11). if the binding is to a site where the nearby site is occupied by another molecule of safranine there is stacking (2224). If the binding is to a site where the nearby site is an empty site, safranine remains in monomeric form. The spectroscopic effects, changes of absorbance and of fluorescence, occur when safranine moves from the monomeric to the dimeric (or higher multimeric) form. The transition involves a large spectral shift with a decrease of absorbance at 520 nm and quenching of fluorescence (11). Vice versa when safranine moves from the dimeric to the monomeric
form there is increase of absorbance and of fluorescence. Presumably during safranine uptake various forms of the dye are simultaneously present depending on the concentration of safranine and on the binding and dimerization constants. Determination of these constants will render the model more quantitative. The extensive binding of safranine to the mitochondrial membrane during A$ driven uptake is in accord with two observations. First, when AlL is calculated on the distribution of safranine between mitochondria and supernatant, values higher than 250 mV may be obtained if an activity coefficient of 1 is assumed (unpublished). Second, under conditions of massive safranine uptake there is a negligible increase of matrix volume (11). Endpoint Titrations The interaction of polyanions with metacromatic dyes has been analyzed with endpoint titrations (22-24). In the range where the number of dye molecules is in excess with respect to the number of binding sites, increase of the number of sites causes increased dye binding and stacking (decrease of absorbance). The stacking reaches its maximum (saturation point) when the number
258
ZANOTTI
FIG. 2. Endpoint titration of safranine with mitochondria at three dye concentrations. The merlium contained 0.2 M sucrose, 20 mM Hepes pH 7.2, 0.1 mM EGTA, 2 mM P,-Tris, 4 pM rotenone, 1 /*g oligomycin x mg prot-‘. The absorbancy changes were obtained by adding, to rotenone-treated mitochondria, either 5 mM succinate-Tris or 90 pmol valinomyxin x mg prot-‘. [K’],) was 0.1 miv. Temperature, 20°C.
of binding sites is equivalent to the number of dyes. Further increase of the number of sites results in a dilution of the dye molecules on nonadjacent sites, with destacking and transition of the dye to the monomeric form. Thus increase of the number of sites in excess to those required to bind all the dye molecules results in destacking due to redistribution of the dyes on distant sites. Colonna et al. (11) have carried out endpoint titrations during respiration-driven dye uptake in rat liver mitochondria. In Fig. 2 the titrations during respiration-driven dye uptake are compared with those obtained during K+ diffusion potential-driven dye uptake at three dye concentrations. The endpoint titrations with mitochondria may be interpreted in a way similar to that for the binding of metachromatic dyes to polyanions in spite of the fact that, in the case of mitochondria, the binding of dyes occurs after a process of translocation and distribution at electrochemical equilibrium of the dye. In both endpoint titrations there is a range where the increase of the amount of mitochondria was accompanied by a parallel
AND AZZONE
larger absorbancy change. In this range there is an excess of dye and therefore the larger absorbancy change accompanying the increase of protein is due to an increase of amount of dye undergoing uptake and stacking. After reaching a maximum, further increase of the amount of protein was accompanied by a reduction of extent of absorbancy change. This is the range of excess of protein. In this range the increase of amount of protein is accompanied by an increase of the amount of dye taken up. However the excess of binding sites leads to decrease of the amount if dye taken up per milligram of protein, and therefore to dye dilution on the binding sites, destacking, and increase of absorbancy. As expected, the amount of protein required for “saturation” was proportional to the dye concentration. There were two differences between endpoint titrations during respiration-driven and diffusion potential-driven dye uptake. First, the extent of absorbance change due to the K+ diffusion potential of 180 mV was, at all protein concentrations, lower than that due to succinate oxidation. This indicates that either the aerobic potential is higher than 180 mV or that calculation of the K+ diffusion potential on the basis of the Nernst equation is not correct. Second, at all three dye concentrations, the “saturation” point was reached at a higher protein concentration when the driving force was the diffusional potential. This is due to the fact that the diffusion potential-driven dye uptake involves an exchange of safranine with matrix K+. At high protein concentrations the amount of K+ efflux required for electrochemical equilibrium of the dye is negligible and does not interfere with the theoretical K+ diffusion potential. At low protein concentrations, on the other hand, the amount of K+ efflux becomes appreciable with respect to the matrix K+. This lowers the K+ diffusion potential and then the safranine response. Figure 2 indicates that calibration of the safranine response with K+ diffusion potentials cannot be carried out at high dyelprotein ratios but only at dye/protein ratios lower than those giving “saturation” in the diffusion potential endpoint titration. At high dye/protein ratios the difference between
SAFRANINE
AS MEMBRANE
POTENTIAL
PROBE
2.59
FIG. 3. Correlation between absorbance changes of safranine and K’ diffusion potential. Experimental conditions as in Fig. 2; 25 pM safranine. The absorbancy changes were obtained adding 90 pmol valinomycin x mg prot-’ to rotenone-treated mitochondria. The [K+],/[K+], ratio was varied by increasing KC1 in the medium. In B, the absorbancy changes are expressed as percentage of the value recorded with a [K+],/[K+]O ratio of 1000. The &!J values reported in the abscissas were calculated by applying the Nernst equation to the [K+]J[K+],, ratio.
the two titrations is partially due to loss of the matrix K+ due to exchange with safranine. Calibration
of the Safrardne
Response
The endpoint titrations curve of Fig. 2 suggests that the calibration curves of the safranine response on the basis of K+ diffusion potential should be affected by the variations of the dye/protein ratios. This point is further analyzed in Figs. 3-6. Figure 3 B shows the absorbance changes of safranine in the presence of increasing potentials. The dye concentration was kept constant, 25 FM, and the amount of protein varied. Two points are relevant. First, the extent of absorbance change decreased markedly with the increase of the amount of protein. This is in accord with the titration of Fig. 2. Second, the safranine response was nonlinear in the high potential region. The deviation from linearity of the calibration curve was a function of the dye/protein ratio, larger deviations occurring at lower ratios. In Fig. 3A the effect of the dyelprotein ratio is examined by plotting the change of absorbance as a percentage of the maximal
absorbance change. The lower the dyelprotein ratio, the larger was the deviation from linearity in the high potential region. Furthermore the lower the dye/protein ratio the larger was the shift of the calibration curve toward lower potentials. As shown in Fig. 4 the extent of linearity of the calibration curve was also a function of the dye concentration at constant dye/ protein ratios. The higher the dye concentration, the larger was the deviation from linearity in the high potential region and the shift of the calibration curve toward lower potentials. The lack of linearity of the calibration curve has already been observed with other potential probes (‘7-9,25-26). The deviation from linearity in the high potential region has been interpreted as due either to the effect of ion leaks or to the intrinsic features of the response of the optical probes. The former explanation has been tested by calculating the K+ diffusion potential with the [3H]triphenylmethy1phosphonium distribution. Figure 5 shows that the A$ based on the K+ distribution was, in the low potential region, identical to that based on the
260
ZANOTTI
AND AZZONE
energized state. Since the half-time for the H+ pump to charge the membrane is very fast the values on the ordinate
reflect the
time required for safranine to reach electrochemical equilibrium. Figure 7A indicates that the half-time
1
50
I
100
Ay.mv
1
150
FIG. 4. Correlation between absorbancechangeof safranine and K+ diffusion potential at constant dye/ protein ratios and increasing dye concentrations. Ex-
perimental conditions as in Fig. 2 except that the amount of protein was varied at eachdye concentration to maintain the dye/protein
ratio constant.
triphenylmethylphosphonium distribution. On the other hand the A$ based on the organic cation distribution was lower than the Nernst potential calculated on the K+ distribution in the high potential region. The deviation is not due to the amount of organic cation uptake (-1 nmol x mg prot-‘) and is attributed to H+ leakage. The data of Fig. 3A have been replotted in Fig. 6 by using as reference the A$ calculated on the distribution of organic cations. The corrected plots of Fig. 6 indicate an
decreased with the in-
crease of mitochondrial protein while Fig. 7B shows that when the dye/protein ratio was kept constant the half-time decreased with the increase of dye concentration. Two factors determine the dimension of the t1,2 for redistribution signal probes: first, the rate of translocation of the probe across the membrane, which in turn depends on the membrane permeability and the concentration of the probe; second, the amount of probe translocation required to reach electrochemical equilibrium. Factor 2 explains the increase or t ,,2 with the increase of dyelprotein ratio. Factor 1, on the other hand, explains the increase of t,,2, at constant dye/ protein ratios, with the decrease of dye concentration. In fact the rate of dye translocation is proportional to the dye concentration (unpublished) according to the general behavioi of hydrophobic ions (27). -
23
,’ Cakuktyi
,,/”
extensive linearity of the safranine response at high dye/protein ratios up to a A$ of
120- 130mV. However, lowering of the dye/ protein ratio was still accompanied by deviation from linearity. Time Response of Safranine
Figures 7A and B shows the time response of safranine. This was measured as t L,2in view of the first-order nature of safranine kinetics (12). The values on the ordinate indicate the half-time for safranine to monitor the transition from the anaerobic to the
Cakuktd
Nmt
potmtiil
FIG. 5. Correlation between dimension of K+ diffusion potential calculated on K’ distribution and measured on the triphenylmethylphosphonium distribution. The theoretical valueswere calculatedby applying the Nernst equation to the [K+]i/[K+]O ratio. The experimental values were measured as described under Experimental. Experimental conditions as in Fig. 2; 2 mg prot/ml. The activity coefficient for triphenylmethylphosphonium was assumed as 0.6.
SAFRANINE
f J
0.2
AS MEMBRANE
B
I/
/
Charge Separation Potential
Ay.mr FIG. 6. Relationship between absorbance changes of safranine and K+ diffusion potential. The values for the safranine absorbance changes were taken from Fig. 3. The values for the K+ diffusion potential were measured with the [3H]triphenylmethylphosphonium distribution.
Inhibitory
261
PROBE
tions higher than 70-80 FM, safranine caused a slight stimulation of the state 4 respiration. This indicates a slight uncoupling effect of safranine. At concentrations higher than 40 pM safranine caused a progressive inhibition of the FCCP-stimulated respiration. This indicates that safranine interferes either with electron transfer or with H+ pumping coupled to electron transfer. The more marked inhibitory effect of safranine was however on the ADP-stimulated respiration. The respiratory control ratio (?ADP respiratory rate) was decreased by safranine even at the lowest concentration. This indicates an inhibitory effect of safranine either at the level of the ATPase or of the adenine nucleotide translocase.
l 0.8mg/h1 01.5 . A30 . racl.
I . 0.l
POTENTIAL
Effects of Safrankne
Figure 8 shows the inhibitory effects of safranine on electron transport, energy coupling, and energy transfer. At concentra-
and Membrane
Figure 9 shows that the rate of safranine response increased linearly with the increase of A+. The experiment is! in accord with the view that the rate of transport of a species moving electrophoretically in a function of the electrical field. The experiment of Fig. 9 predicts that the rate of maximal safranine response in aerobic mitochondria corresponds to the establishment of a steady state AI/J. In Fig. 10 safranine has been used to monitor the number of charges required to build a steady state potential after an oxidant
B I
1
I
1 2 3 mcKhmdrWproWn,rnqAnl 1
i
** tin**.
p
FIG. 7. Half-time of safranine response after addition of succinate to rotenone-treated mitochondria. Experimental conditions as in Fig. 2. In A, 25 pM safranine and increasing amounts of protein. In B, the amount of safranine was varied and the dye/protein ratio kept constant at the level of 20.
262
ZANOTTI
AND AZZONE
FIG. 8. Inhibitory effects of safranine on mitochondria. Experimental conditions as in Fig. 2, 5 mu succinate. The respiratory rate was measured in the absence and presence of 2 pM FCCP or of 200 pM ADP; 1 mg prot/ml.
pulse. Increasing amounts of oxygen were added to anaerobic mitochondria incubated in the presence of succinate and the rate at which the safranine absorbance decreased after initiation of the respiration was determined. Establishment of steady state potential was obtained at about 0.5 natom oxygen
x mg prot-I. Fifty percent of the maximal rate was obtained at about 0.15 natom oxygen X mg prot’. It is important to note that the values obtained in Fig. 10 represent only an upper limit since diffusion of safranine across the membrane results in collapse of A$ and therefore in increase of the amount of charge separation required to build up the potential. However under the conditions of Fig. 10 the rate of safranine uptake is more than one order of magnitude slower than the rate of H+ extrusion calculated on the state 4 respiratory rate. In fact the state 4 respiratory rate is not modified during the uptake of 20 PM safranine.
DISCUSSION
Akerman and Wikstrom (10) have proposed the permeant cation cyanine dye safranine 0 to monitor transmembrane
FIG. 9. Relationship between rate of safranine uptake and K+ diffusion potential. Experimental conditions as in Fig. 2. Safranine, 20 pM, 2 mg prot/ml. The absorbancy changes were obtained adding 90 pmol valinomycin x mg prott’ to rotenone-treated mitochondria. The [K+li/[KfJ, ratio was varied by increasing KCI in the medium.
po-
tential in mitochondria and other organelles. The present study also indicates a qualitative correlation between A+ and safranine response although it raises a number of problems as to the quantitative use of the safranine response for the determination of A$. The first problem is the relationship between extent of safranine absorbance change and amount of mitochondrial protein. The higher the amount of protein the lower is the extent of absorbance change at equivalent changes of A+ This is due to the fact that most of the absorbance change is due
SAFRANINE
AS MEMBRANE
to stacking of safranine on membrane sites. An increase of protein results in an increased probability that dye binding occurs to sites where the nearby site is not occupied by another dye molecule. In this case the dye remains in a monomeric form, and the dye uptake is not accompanied by changes in absorbance. The second problem is the linearity of the dye response to AI,!I changes. While other “redistribution signal” probes do not show linearity (13), Akerman and Wikstriim (10) have reported a calibration curve with a linear response of safranine in the range between 50 and 170 mV. In the present study we find that the response is nonlinear at low dye/protein ratios and approaches linearity at high dye/protein ratios. The nonlinearity is partly due to the fact that in the high potential region, the A$ calculated by applying the Nernst equation to the K+ distribution seems not to correspond to the A$ calculated on the distribution of organic cations. The diffusion potential approaches the Nernst potential only when the permeability for K’ is much higher than for other ions. The discrepancy between A$ calculated on I<+ and organic cation distribution is presumably due to H+ leaks. A more fundamental reason for the nonlinearity seems however to reside in the nature of the safranine response as redistribution signal probe. Tsien and Hladky (28) have shown that the nonlinearity of the 3,3’-dipropylthiodicarbocyanine, di-S-C, (5) response in red cells is due to a “saturation effect.” When most of dye is already taken up by the red cell, a further increase of A$ results only in a negligible further uptake of dye and then in miniscule absorbance change. The experimental values obtained during uptake of di-S-C, (5) in red cells are in good agreement with the predictions of a model, not dissimilar from that of Fig. 1. A saturation effect in mitochondria is supported by the dependence of the linearity of dye response on the dye/protein ratio. At all dye/protein ratios there is a region at which the dye response is linear (Fig. 3). However the lower the dye/protein ratio the lower is the value of A$ at which the dye response becomes nonlinear. Being that safranine is distributed at electrochemical
POTENTIAL
PROBE
263
FIG. 10. Initial rate of safranine uptake after addition of increasing amount of oxygen to anaerobic mitochondria. Experimental conditions as in Fig. 2. Safranine, % FM, 5 mM succinate, 2 mg protiml. Increasing amount of oxygen, as indicated in the abscissa, were added to an anaerobic mitochondrial suspension.
equilibrium, the lower the dye/protein ratio the higher is the amount of dye taken up by the mitochondria at equivalent values of A$. Thus the “saturation point” is reached at lower A$ values when the dye/protein ratio is low. A similar reasoning explains also why at constant dye/protein ratios, the lower the dye concentration, the higher is the value of A$ at which the deviation from linearity initiates. In conclusion our data indicate that the linearity of the safranine response increases with the increase of dye/ protein ratios. However at high dye/protein ratios calibration of the safranine response with I(+ diffusion potentials is hindered by the amount of loss of matrix K+ in exchange with safranine. A third problem is the time response of the dye. As shown in Fig. 7 the relationship between tliZ and amounts of protein or of dye is hyperbolical. This is not unexpected for a probe monitoring,A$ by redistributing across the membrane since this renders the t ,,p dependent on the rate and amount of dye transport across the membrane. The tlr2 of the dye response remains in the range of 3-5 s below a dye/protein ratio of 12. At higher dye/protein ratios the t1,2 increases considerably, and is higher than 20 s above a dye/protein ratio of 50. There is an inverse correlation between conditions for linearity of dye response and for decrease oftliZ, since the faster probe response occurs at the low dye/protein ratios where there is a more marked deviation from linearity. This represents a limit to the utilization of the dye.
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AND AZZONE
A fourth problem is the toxic effects of the dye, expected for any extrinsic probe above certain concentrations. Figure 8 shows that the uptake of safranine leads to inhibition of electron transport, of energy coupling, and of energy transfer. However these effects occur at different dye concentrations. The effect on energy coupling is negligible since, at a dyeiprotein ratio of 80, under conditions where almost all the dye is inside the mitochondria and bound to the membrane, the stimulation of the state 4 respiration is about 10%. More marked is the effect on the respiratory rate since 50% inhibition of the FCCP-stimulated respiration occurs at a dye/protein ratio of 150, but under conditions where the dye uptake is negligible. The more marked effect of the dye appears to consist in abolishing the ADP-induced stimulation of respiration. Both the inhibition of e- transfer and of ADP induced respiration are consistent with the view that the dye, a lipophilic cation, acts as an inhibitor of the H+ channels, both at the level of the redox complex and of the ATPase. Effects similar to those observed here with safranine have been reported for other extrinsic probes acting as permeant lipophilic cations and thus presumably taken up, accumulated and bound to the membrane (8, 30, 31). This suggests that the toxic effects of safranine are nonspecific. The experiment of Fig. 10 is the first attempt to determine the amount of oxygen uptake required to charge the mitochondrial membrane, during succinate oxidation, at the level of the static head potential. The dimension of the electrical field is followed on the basis of the rate of safranine uptake. We show in Fig. 9 that this rate increases linearly with AI/J. Hence the maximal rate of safranine uptake corresponds to the maximal AJI existing in mitochondria which is, by definition, that found under static head conditions. The minimal amount of oxygen to reach the maximal rate of safranine uptake then becomesthe amount of respiration required to induce a metabolic transition from anaerobiosis to energization, i.e., the extent of charge separation, due to the redox H+ pump, to increase A$ from zero to the static head level. This amount of oxygen is about 0.5 natom x mg prot-’ correspond-
ing, on the basis of stoichiometry of 8 H+/O, to the extrusion of 4 nmol H+ x mg prot-‘. This value is in reasonable agreement with the calculation made by Mitchell (29) of 1 nmol H+ x mg prot- I. The agreement between experiment and prediction indicates that the two values on which the prediction was based are presumably correct: (i) a mitochondrial surface of 40 m’lg prot; (ii) a capacitance of the mitochondrial membrane of 1 pF/cm2. The low amount of oxygen uptake required to raise A$ to the steady state level explains the requirement for counter ions for any microscopic ion flux in mitochondria. This requirement permits the replacement of H+ with cations for any H+ pump stoichiometry measured in steady state, i.e., involving an amount of oxygen uptake greater than 0.2 natom oxygen x mg prot-’ . The above considerations however should not underestimate a fundamental discrepancy. While 0.5 natom x oxygen x mg prot-’ is required to charge the membrane, protons are extruded in the presence but not in the absence of permeant cations (3234). Why protons are not seen after oxygen pulses in the absence of permeant cations is obscure. A reasonable explanation is that the basic event in energy transduction is the formation of an electrical field within the membrane. This is followed by H+ extrusion in the outer aqueous phase only when permeant cations move across the membrane down the electrical field. REFERENCES 1. ROTTENBERG, H. (1975) J. Bioenerg. 7, 61-74. 2. AZZONE, G. F., BRAGADIN, M., POZZAN, T., AND DELL’ANTONE, P. (1976) Biochim. Biophys. Acta 459, 96- 109. 3. AZZI, A., GHERARDINI, P., AND SANTATO, M. (1971) J. Biol. Chem. 246,2035-2042. 4. JASAITIS, A. A., KULIENE, V. V., AND SKULACHEV, V. P. (19’71) Biochim. Biophys. Acta 234, 177- 181. 5. RADDA, G. K., AND VANDERKCMX, J. (1972) Biochim. Biophys. Acta 265, 509-549. 6. FERGUSON, S. J., LLOYD, W. S., AND RADDA, G. K. (1976) Btichim. Biophys. Acta 423, 174- 188. 7. LARIS, P. C., BAHR, P. D., ANDCHAFFEE, R. R. J. (1975) Biochim. Biophys. Acta 376,415- 425.
SAFRANINE
AS MEMBRANE
8. KINNALLY, W. K., TEDESCHI, H., AND MALOFF, B. L. (1978) Biochemistry 17,3419-3428. 9. KINNALLY, W. K., AND TEDESCHI, H. (1976) FEBS Lett. 62, 41-45. 10. AKERMAN, K. E. O., AND WIKSTROM, M. F. (1976) FEBS Lett. 68, 191-197. 11. COLONNA, R., MASSARI, S., AND AZZONE, G. F. (1973) Eur. J. Biochem. 34, 577-585. 12. AKERMAN, K. E. O., AND SARIS, N. E. L. (1976) Biochim. Biophys. Acta 426,624-629. 13. COHEN, L. B., AND SALZBERG, B. M. (1978) Rev. Physiol. Biochem. Pharrnacol. 83, 36-88. 14. ZANOTTI, A., AND AZZONE, G. F. (1979) in XIth lnternational Congress of Biochemistry, Toronto, Canada. 15. MASSARI, S., BALBONI, E., AND AZZONE, G. F. (1972) Biochim. Biophys. Acta 283, 16-22. 16. NICHOLLS, D. G. (1974) Eur. J. Biochem. 50, 305-315. 17. AZZONE, G. F., POZZAN, T., MASSARI, S., AND BRAGADIN, M. (1978) Biochim. Biophys. Acta 501, 296-306. 18. AZZONE, G. F., POZZAN, T., AND MASSARI, S. (1978). Biochcm. Biophys. Acta 501, 307-316. 19. ROSSI, E., AND AZZONE, G. F. (1969) Eur. J. Biochem. 7, 418-426. 19a. BRAGADIN, M., POZZAN, M., ANDAZZONE, G. F. (1979) Biochemistry 18, 5972-5978. 20. PAL, M. K., AND SCHUBERT, M. (1962) J. Amer. Cherrr. Sot. 84. 4384-4393.
POTENTIAL
PROBE
265
21. WAGGONER, A. S. (1976) J. Membrane Biol. 27, 317-334. 22. STONE, A. L., AND BRADLEY, D. F. (1961) J. Amer. Chem. Sot. 83, 3627-3634. 23. STONE, A. L., AND BRADLEY, D. F. (1967) Biochim. Biophys. Acta 148, 172-192. 24. VITAGLIANO, V., AND COSTANTINO, L. (1970) J. Phys. Chem. 74, 197-202. 25. HLADKY, S. B., AND RINK, T. J. (1976)J. Physiol. 263, 287-319. 26. HOFFMANN, J. F., AND LARIS, P. C. (1974) J. Physiol. 239, 519-552. 27. KETTERER, B., NEUMCKE, B., AND LAOGER, P. (1971) J. Membrane Biol. 5, 225-245. 28. TSIEN, R. Y., AND HLADKY, S. B. (1978)d. Merrcbrane Biol. 38, 73-97. 29. MITCHELL, P. (1966) Biol. Rev. 41,445-502. 30. HIGUTI, T., YOKOTA, M., ARAKAKI, N., HATTORI, A., AND TANI, I. (1978) Biochfim. Biophys. Acta 503, 211-222. 31. GRIMWOOD, B. G., AND WAGNER, R. P. (197(i) Arch. Biochem. Biophys. 176, 43-52. 32. CHAPPELL, B. J., AND HAARHOFF, K. (1967) i?l The Biochemistry of Mitochondria (Slater, E. C., et al., eds.), pp. 75-91, Academic Press, New York/London. 33. CHANCE, B., AND MELA, L. (1966) Nature (LOW don) 212, 372-376, 34. POZZAN, T., AND AZZONE, G. F. (1976) FEBS Lett. 71, 62-66.
Safranine
as Membrane Potential Rat Liver Mitochondria
Probe in
ADRIAN0 ZANOTTI AND GIOVANNI FELICE AZZONE With the technical assistance of Paolo Veronese C.N.R.
lJnit,for
the Study of Physiology sf Mitochondria and Institzlte University qf Padua, Italy Received October 18, 1979; revised December
qfGexera1
Pathology,
11, 1979
The absorbance changes accompanying safranine uptake due either to respiration or to K+ diffusion potential are maximal at a point of equivalence between number ofdye molecules and binding sites (“saturation” point) and decrease both below and above this point. This behavior is in accord with a model where safranine uptake is followed by stacking on adjacent membrane sites below saturation and destacking on nonadjacent sites above saturation. The values of the endpoint titrations are similar for the respiration- and K’ diffusion potentialinduced safranine uptakes only at protein concentrations above saturation. Calibration of the safranine response with K+ diffusion potentials indicates a linear region at low AI/J and a deviation from linearity at high &/I. The region of linear safranine response depends on the dye/protein ratio. Increase of dye/protein ratio leads to expansion of the linear range. However calibration at high dye/protein ratios is limited by the loss of matrix K+. At constant dye/protein ratios increase of dye concentration shifts the calibration curve at lower & values. The half-time of the safranine response increases hyperbolically with the decrease of protein at constant dye concentration and with the decrease of dye concentration at constant dye/protein ratios. Uptake and binding of safranine result in mitochondrial damage with respect to degree of energy coupling, rate of electron transport, and ADP-stimulated respiration. Oxygen pulses to anaerobic mitochondria result in an electrical field which may be followed with the safranine response. The minimal amount of oxygen capable of inducing a maximal rate of safranine uptake is 0.5 natom x mg protein’. Since the rate of safranine uptake is linear with A+, this amount of oxygen corresponds to the extent of charge separation required for a steady state A$.
electrode impalement technique, optical probes are the natural candidates. Azzi et al. (3) and Jasaitis et al. (4) have proposed the use of ANS.’ The extensive disagreement in interpreting the fluorescent response of ANS has however considerably limited the application of this probe (5, 6). Cyanine dyes have been used by Laris (7) and by Kinally et aZ. (8, 9). A coherent picture on the behavior of these probes seems to emerge from the studies of Laris (cf. however Ref. (8)). Akerman and WikStrom (10) have reported a linear response
It is now generally accepted that in steady state a large H+ electrochemical gradient exists across the membrane of energy-transducing organelles and that this H+ electrochemical gradient (AWL.,) drives various energy-requiring reactions. Since the major component of A& is the membrane potential (A$) a number of techniques have been developed for the determination of A+. The most widely used procedures are based on the distribution of permeant ions (1,2) and they are thought to provide reliable indications of A$ under steady state conditions. The inadequacy of the radioisotope distribution method for the case of short transients has however prompted interest toward alternative approaches. Because of the problems arising from the use of the
’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)LpiperazineethanesuIfonic acid; EGTA, ethylene glycol bis(@aminoethyl ether)N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; prot, protein; ANS, 1 anilinonaphtalene-8sulphonate; FCCP, carbonylcyanide-ptrifluoromethoxyphenylhydrazone. 255
0003-9861/80/050255-11$02.00/0 Copyright G 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.
256
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AND AZZONE
of safranine, with respect to AI/I, due to K+ and H+ diffusion. This dye had been previously shown to undergo accumulation and stacking in mitochondria during respiration (11) and in liposomes during K+ efflux (12). Safranine is a “redistribution signal” probe (13) since its spectroscopic response is due to its redistribution at the two sides of the membrane under the effect of A$. In order to test the validity of safranine as A$ probe we have investigated the linearity of the safranine response as a function of A$, and the sensitivity and time course of the response. Furthermore since the probe is a lipophilic molecule its damaging effect on the membrane has been tested. Once ascertained as to the advantages and limitations of safranine as A$ probe, we have used safranine to investigate the relationship between charge separation and rise of A$. A preliminary report of the present study has already been presented (14). EXPERIMENTAL Rat liver mitochondria were prepared as described previously (15). The last washing was carried out in an EDTA-free medium. The standard incubation medium contained 0.2 M sucrose, 20 mM Hepes pH 7.2, 2 mM phosphate-Tris, 0.1 mM EGTA-Tris, 4 PM rotenone. Respiration was initiated by the addition of 5 mM succinate. Absorbancy changes were recorded with a dual-wavelength spectrophotometer (Aminco DW2 a) at the wavelengths 520-554 nm. The net absorbance changes of safranine were calculated by taking as a reference the absorbance of a mitochondrial suspension containing the same amount of protein and the same dye concentration and supplemented with 2 pM FCCP, 80 pmol valinomycin x mgproteinl, and 50 mM KCl. These experimental conditions were selected in order to minimize the Donnan potential in anaerobicuncoupled mitochondria (10, 16). The rate of oxygen uptake was determined with a Clark electrode in a thermostated cuvette. The dimension of A+ was measured with PH]triphenylmethylphosphonium in the following manner (2, 17, 18). The mitochondrial suspension was supplemented with the radioisotopes and centrifuged after 60 s in the S12 rotor of a Sorvall RC2B superspeed centrifuge. The mitochondrial pellet was dissolved at 40°C in 10% SDS, 0.01 M NaOH, 1% mercaptoethanol, and then added to the Packard emulsifier scintillator special MI-96. The distribution of the radioactivity between pellet and supernatant was then determined in a Packard Tri-Carb 2455 liquid scintillation spectrometer. The concentration of triphenylmethylphosphonium
in the matrix was calculated by assuming a volume of 0.5 pl/mg prot-I. Rate and extents of K+ efflux during safranine equilibration were determined with the KS electrode in order to calculate exactly the K+ diffusion potential on the K+ distribution. In the presence of 100 pM [K+], the rates of K’ efflux were 13.6, 21.8, 25.4, and 30.4 nmol K+ x mg prott’ x min-I, and the extent of K+ efflux 18.0, 21.8, 24.4, and 27.5 nmol x mg prott’ at dye/protein ratios of 5, 10,20, and 40, respectively. These data support the view that, except at very high dye/protein ratios and very high values of A$, use of the values of [K’], and [K’], at the moment of the addition of valinomycin introduces a negligible error in the calculation of A$. This further follows from two considerations. First, although the initial [K+], is generally assumed as 100 mM (19), the total extent of K+ efflux in the presence of valinomycin + uncouplers is only 30 nmol X mg pro?. However K+ efflux is accompanied by osmotic shrinkage. This reduces the matrix volume and maintains [K+] relatively constant as indicated by the linearity of the rate of K+ diffusion-driven Ca2+ uptake for an extended period (19a). Second, above 300 FM [K+],, K+ efflux increases [K+], only of a few micromolar, as compared to the initial values, with a drop in AJ, of less than 4 mV. As will be shown in Fig. 5, the A+ calculated on the K+ distribution was compared with that calculated on the distribution of triphenylmethylphosphonium. In the oxygen pulse experiments the medium was bubbled with pure nitrogen. The cuvette was then covered with paraffin oil to restrict oxygen diffusion. Residual traces of oxygen were removed by letting the mitochondria stay in the parafflin oil-covered cuvette for 10 min before oxygen addition. The oxygen diffusion through the paraffin layer, from the air to the oxygendepleted medium, was measured with oxygen electrode and found to be lower than 2.5 pM 0, x min+. Mitochondrial protein was determined with the biuret using bovine serum albumin as a standard. The term dye/protein ratio in the text refers always to nanomoles of dye x milligrams of protein-‘. Safranine was a product of BDH Chemicals Ltd., purified according to Pal and Schubert (20). Aliquots were taken from a standard solution at a concentration of 5 mM. rH]Triphenylmethylphosphonium was a kind gift of Dr. R. Kabach. All other products were of the maximal purity commercially available. RESULTS
The Interaction
Mitochondrial
of Safranine Membrane
with the
The model analyzed in Fig. 1 stems from the evidence that the spectroscopic response of “redistribution signal” probes in general, and of safranine in particular, is correlated
SAFRANINE
AS MEMBRANE
+ + + + + + + +
POTENTIAL
PROBE
257
+ + + + + F + + + A
FIG. 1. Uptake and interaction of safranine with the mitochondrial membrane. “S,, free safranine as monomer; “Sv, free safranine as tlimer or higher multimers; ‘IS,,. bound safranine as monomer; ‘S,, bound safranine in stacked form. The line AB represents the line along which zT-electrons may be shared.
with aggregation or “stacking” phenomena (cf. Refs. (11, 13, 21)) after accumulation of the dye. Safranine moves from the outer to the inner space under the influence of A$ and distributes at electrochemical equilibrium, external >‘S, + matrix zlSr. The concentration of free safranine in the matrix reaches, in presence of a A$ of 120- 180mV, negative inside, the millimolar range. Part of safranine undergoes aggregation in solution, i.e., formation of dimers or of higher multimers. Part of safranine undergoes binding to membrane anionic sites. These anionic sites appear to be located in a hydrophobic environment (11). if the binding is to a site where the nearby site is occupied by another molecule of safranine there is stacking (2224). If the binding is to a site where the nearby site is an empty site, safranine remains in monomeric form. The spectroscopic effects, changes of absorbance and of fluorescence, occur when safranine moves from the monomeric to the dimeric (or higher multimeric) form. The transition involves a large spectral shift with a decrease of absorbance at 520 nm and quenching of fluorescence (11). Vice versa when safranine moves from the dimeric to the monomeric
form there is increase of absorbance and of fluorescence. Presumably during safranine uptake various forms of the dye are simultaneously present depending on the concentration of safranine and on the binding and dimerization constants. Determination of these constants will render the model more quantitative. The extensive binding of safranine to the mitochondrial membrane during A$ driven uptake is in accord with two observations. First, when AlL is calculated on the distribution of safranine between mitochondria and supernatant, values higher than 250 mV may be obtained if an activity coefficient of 1 is assumed (unpublished). Second, under conditions of massive safranine uptake there is a negligible increase of matrix volume (11). Endpoint Titrations The interaction of polyanions with metacromatic dyes has been analyzed with endpoint titrations (22-24). In the range where the number of dye molecules is in excess with respect to the number of binding sites, increase of the number of sites causes increased dye binding and stacking (decrease of absorbance). The stacking reaches its maximum (saturation point) when the number
258
ZANOTTI
FIG. 2. Endpoint titration of safranine with mitochondria at three dye concentrations. The merlium contained 0.2 M sucrose, 20 mM Hepes pH 7.2, 0.1 mM EGTA, 2 mM P,-Tris, 4 pM rotenone, 1 /*g oligomycin x mg prot-‘. The absorbancy changes were obtained by adding, to rotenone-treated mitochondria, either 5 mM succinate-Tris or 90 pmol valinomyxin x mg prot-‘. [K’],) was 0.1 miv. Temperature, 20°C.
of binding sites is equivalent to the number of dyes. Further increase of the number of sites results in a dilution of the dye molecules on nonadjacent sites, with destacking and transition of the dye to the monomeric form. Thus increase of the number of sites in excess to those required to bind all the dye molecules results in destacking due to redistribution of the dyes on distant sites. Colonna et al. (11) have carried out endpoint titrations during respiration-driven dye uptake in rat liver mitochondria. In Fig. 2 the titrations during respiration-driven dye uptake are compared with those obtained during K+ diffusion potential-driven dye uptake at three dye concentrations. The endpoint titrations with mitochondria may be interpreted in a way similar to that for the binding of metachromatic dyes to polyanions in spite of the fact that, in the case of mitochondria, the binding of dyes occurs after a process of translocation and distribution at electrochemical equilibrium of the dye. In both endpoint titrations there is a range where the increase of the amount of mitochondria was accompanied by a parallel
AND AZZONE
larger absorbancy change. In this range there is an excess of dye and therefore the larger absorbancy change accompanying the increase of protein is due to an increase of amount of dye undergoing uptake and stacking. After reaching a maximum, further increase of the amount of protein was accompanied by a reduction of extent of absorbancy change. This is the range of excess of protein. In this range the increase of amount of protein is accompanied by an increase of the amount of dye taken up. However the excess of binding sites leads to decrease of the amount if dye taken up per milligram of protein, and therefore to dye dilution on the binding sites, destacking, and increase of absorbancy. As expected, the amount of protein required for “saturation” was proportional to the dye concentration. There were two differences between endpoint titrations during respiration-driven and diffusion potential-driven dye uptake. First, the extent of absorbance change due to the K+ diffusion potential of 180 mV was, at all protein concentrations, lower than that due to succinate oxidation. This indicates that either the aerobic potential is higher than 180 mV or that calculation of the K+ diffusion potential on the basis of the Nernst equation is not correct. Second, at all three dye concentrations, the “saturation” point was reached at a higher protein concentration when the driving force was the diffusional potential. This is due to the fact that the diffusion potential-driven dye uptake involves an exchange of safranine with matrix K+. At high protein concentrations the amount of K+ efflux required for electrochemical equilibrium of the dye is negligible and does not interfere with the theoretical K+ diffusion potential. At low protein concentrations, on the other hand, the amount of K+ efflux becomes appreciable with respect to the matrix K+. This lowers the K+ diffusion potential and then the safranine response. Figure 2 indicates that calibration of the safranine response with K+ diffusion potentials cannot be carried out at high dyelprotein ratios but only at dye/protein ratios lower than those giving “saturation” in the diffusion potential endpoint titration. At high dye/protein ratios the difference between
SAFRANINE
AS MEMBRANE
POTENTIAL
PROBE
2.59
FIG. 3. Correlation between absorbance changes of safranine and K’ diffusion potential. Experimental conditions as in Fig. 2; 25 pM safranine. The absorbancy changes were obtained adding 90 pmol valinomycin x mg prot-’ to rotenone-treated mitochondria. The [K+],/[K+], ratio was varied by increasing KC1 in the medium. In B, the absorbancy changes are expressed as percentage of the value recorded with a [K+],/[K+]O ratio of 1000. The &!J values reported in the abscissas were calculated by applying the Nernst equation to the [K+]J[K+],, ratio.
the two titrations is partially due to loss of the matrix K+ due to exchange with safranine. Calibration
of the Safrardne
Response
The endpoint titrations curve of Fig. 2 suggests that the calibration curves of the safranine response on the basis of K+ diffusion potential should be affected by the variations of the dye/protein ratios. This point is further analyzed in Figs. 3-6. Figure 3 B shows the absorbance changes of safranine in the presence of increasing potentials. The dye concentration was kept constant, 25 FM, and the amount of protein varied. Two points are relevant. First, the extent of absorbance change decreased markedly with the increase of the amount of protein. This is in accord with the titration of Fig. 2. Second, the safranine response was nonlinear in the high potential region. The deviation from linearity of the calibration curve was a function of the dye/protein ratio, larger deviations occurring at lower ratios. In Fig. 3A the effect of the dyelprotein ratio is examined by plotting the change of absorbance as a percentage of the maximal
absorbance change. The lower the dyelprotein ratio, the larger was the deviation from linearity in the high potential region. Furthermore the lower the dye/protein ratio the larger was the shift of the calibration curve toward lower potentials. As shown in Fig. 4 the extent of linearity of the calibration curve was also a function of the dye concentration at constant dye/ protein ratios. The higher the dye concentration, the larger was the deviation from linearity in the high potential region and the shift of the calibration curve toward lower potentials. The lack of linearity of the calibration curve has already been observed with other potential probes (‘7-9,25-26). The deviation from linearity in the high potential region has been interpreted as due either to the effect of ion leaks or to the intrinsic features of the response of the optical probes. The former explanation has been tested by calculating the K+ diffusion potential with the [3H]triphenylmethy1phosphonium distribution. Figure 5 shows that the A$ based on the K+ distribution was, in the low potential region, identical to that based on the
260
ZANOTTI
AND AZZONE
energized state. Since the half-time for the H+ pump to charge the membrane is very fast the values on the ordinate
reflect the
time required for safranine to reach electrochemical equilibrium. Figure 7A indicates that the half-time
1
50
I
100
Ay.mv
1
150
FIG. 4. Correlation between absorbancechangeof safranine and K+ diffusion potential at constant dye/ protein ratios and increasing dye concentrations. Ex-
perimental conditions as in Fig. 2 except that the amount of protein was varied at eachdye concentration to maintain the dye/protein
ratio constant.
triphenylmethylphosphonium distribution. On the other hand the A$ based on the organic cation distribution was lower than the Nernst potential calculated on the K+ distribution in the high potential region. The deviation is not due to the amount of organic cation uptake (-1 nmol x mg prot-‘) and is attributed to H+ leakage. The data of Fig. 3A have been replotted in Fig. 6 by using as reference the A$ calculated on the distribution of organic cations. The corrected plots of Fig. 6 indicate an
decreased with the in-
crease of mitochondrial protein while Fig. 7B shows that when the dye/protein ratio was kept constant the half-time decreased with the increase of dye concentration. Two factors determine the dimension of the t1,2 for redistribution signal probes: first, the rate of translocation of the probe across the membrane, which in turn depends on the membrane permeability and the concentration of the probe; second, the amount of probe translocation required to reach electrochemical equilibrium. Factor 2 explains the increase or t ,,2 with the increase of dyelprotein ratio. Factor 1, on the other hand, explains the increase of t,,2, at constant dye/ protein ratios, with the decrease of dye concentration. In fact the rate of dye translocation is proportional to the dye concentration (unpublished) according to the general behavioi of hydrophobic ions (27). -
23
,’ Cakuktyi
,,/”
extensive linearity of the safranine response at high dye/protein ratios up to a A$ of
120- 130mV. However, lowering of the dye/ protein ratio was still accompanied by deviation from linearity. Time Response of Safranine
Figures 7A and B shows the time response of safranine. This was measured as t L,2in view of the first-order nature of safranine kinetics (12). The values on the ordinate indicate the half-time for safranine to monitor the transition from the anaerobic to the
Cakuktd
Nmt
potmtiil
FIG. 5. Correlation between dimension of K+ diffusion potential calculated on K’ distribution and measured on the triphenylmethylphosphonium distribution. The theoretical valueswere calculatedby applying the Nernst equation to the [K+]i/[K+]O ratio. The experimental values were measured as described under Experimental. Experimental conditions as in Fig. 2; 2 mg prot/ml. The activity coefficient for triphenylmethylphosphonium was assumed as 0.6.
SAFRANINE
f J
0.2
AS MEMBRANE
B
I/
/
Charge Separation Potential
Ay.mr FIG. 6. Relationship between absorbance changes of safranine and K+ diffusion potential. The values for the safranine absorbance changes were taken from Fig. 3. The values for the K+ diffusion potential were measured with the [3H]triphenylmethylphosphonium distribution.
Inhibitory
261
PROBE
tions higher than 70-80 FM, safranine caused a slight stimulation of the state 4 respiration. This indicates a slight uncoupling effect of safranine. At concentrations higher than 40 pM safranine caused a progressive inhibition of the FCCP-stimulated respiration. This indicates that safranine interferes either with electron transfer or with H+ pumping coupled to electron transfer. The more marked inhibitory effect of safranine was however on the ADP-stimulated respiration. The respiratory control ratio (?ADP respiratory rate) was decreased by safranine even at the lowest concentration. This indicates an inhibitory effect of safranine either at the level of the ATPase or of the adenine nucleotide translocase.
l 0.8mg/h1 01.5 . A30 . racl.
I . 0.l
POTENTIAL
Effects of Safrankne
Figure 8 shows the inhibitory effects of safranine on electron transport, energy coupling, and energy transfer. At concentra-
and Membrane
Figure 9 shows that the rate of safranine response increased linearly with the increase of A+. The experiment is! in accord with the view that the rate of transport of a species moving electrophoretically in a function of the electrical field. The experiment of Fig. 9 predicts that the rate of maximal safranine response in aerobic mitochondria corresponds to the establishment of a steady state AI/J. In Fig. 10 safranine has been used to monitor the number of charges required to build a steady state potential after an oxidant
B I
1
I
1 2 3 mcKhmdrWproWn,rnqAnl 1
i
** tin**.
p
FIG. 7. Half-time of safranine response after addition of succinate to rotenone-treated mitochondria. Experimental conditions as in Fig. 2. In A, 25 pM safranine and increasing amounts of protein. In B, the amount of safranine was varied and the dye/protein ratio kept constant at the level of 20.
262
ZANOTTI
AND AZZONE
FIG. 8. Inhibitory effects of safranine on mitochondria. Experimental conditions as in Fig. 2, 5 mu succinate. The respiratory rate was measured in the absence and presence of 2 pM FCCP or of 200 pM ADP; 1 mg prot/ml.
pulse. Increasing amounts of oxygen were added to anaerobic mitochondria incubated in the presence of succinate and the rate at which the safranine absorbance decreased after initiation of the respiration was determined. Establishment of steady state potential was obtained at about 0.5 natom oxygen
x mg prot-I. Fifty percent of the maximal rate was obtained at about 0.15 natom oxygen X mg prot’. It is important to note that the values obtained in Fig. 10 represent only an upper limit since diffusion of safranine across the membrane results in collapse of A$ and therefore in increase of the amount of charge separation required to build up the potential. However under the conditions of Fig. 10 the rate of safranine uptake is more than one order of magnitude slower than the rate of H+ extrusion calculated on the state 4 respiratory rate. In fact the state 4 respiratory rate is not modified during the uptake of 20 PM safranine.
DISCUSSION
Akerman and Wikstrom (10) have proposed the permeant cation cyanine dye safranine 0 to monitor transmembrane
FIG. 9. Relationship between rate of safranine uptake and K+ diffusion potential. Experimental conditions as in Fig. 2. Safranine, 20 pM, 2 mg prot/ml. The absorbancy changes were obtained adding 90 pmol valinomycin x mg prott’ to rotenone-treated mitochondria. The [K+li/[KfJ, ratio was varied by increasing KCI in the medium.
po-
tential in mitochondria and other organelles. The present study also indicates a qualitative correlation between A+ and safranine response although it raises a number of problems as to the quantitative use of the safranine response for the determination of A$. The first problem is the relationship between extent of safranine absorbance change and amount of mitochondrial protein. The higher the amount of protein the lower is the extent of absorbance change at equivalent changes of A+ This is due to the fact that most of the absorbance change is due
SAFRANINE
AS MEMBRANE
to stacking of safranine on membrane sites. An increase of protein results in an increased probability that dye binding occurs to sites where the nearby site is not occupied by another dye molecule. In this case the dye remains in a monomeric form, and the dye uptake is not accompanied by changes in absorbance. The second problem is the linearity of the dye response to AI,!I changes. While other “redistribution signal” probes do not show linearity (13), Akerman and Wikstriim (10) have reported a calibration curve with a linear response of safranine in the range between 50 and 170 mV. In the present study we find that the response is nonlinear at low dye/protein ratios and approaches linearity at high dye/protein ratios. The nonlinearity is partly due to the fact that in the high potential region, the A$ calculated by applying the Nernst equation to the K+ distribution seems not to correspond to the A$ calculated on the distribution of organic cations. The diffusion potential approaches the Nernst potential only when the permeability for K’ is much higher than for other ions. The discrepancy between A$ calculated on I<+ and organic cation distribution is presumably due to H+ leaks. A more fundamental reason for the nonlinearity seems however to reside in the nature of the safranine response as redistribution signal probe. Tsien and Hladky (28) have shown that the nonlinearity of the 3,3’-dipropylthiodicarbocyanine, di-S-C, (5) response in red cells is due to a “saturation effect.” When most of dye is already taken up by the red cell, a further increase of A$ results only in a negligible further uptake of dye and then in miniscule absorbance change. The experimental values obtained during uptake of di-S-C, (5) in red cells are in good agreement with the predictions of a model, not dissimilar from that of Fig. 1. A saturation effect in mitochondria is supported by the dependence of the linearity of dye response on the dye/protein ratio. At all dye/protein ratios there is a region at which the dye response is linear (Fig. 3). However the lower the dye/protein ratio the lower is the value of A$ at which the dye response becomes nonlinear. Being that safranine is distributed at electrochemical
POTENTIAL
PROBE
263
FIG. 10. Initial rate of safranine uptake after addition of increasing amount of oxygen to anaerobic mitochondria. Experimental conditions as in Fig. 2. Safranine, % FM, 5 mM succinate, 2 mg protiml. Increasing amount of oxygen, as indicated in the abscissa, were added to an anaerobic mitochondrial suspension.
equilibrium, the lower the dye/protein ratio the higher is the amount of dye taken up by the mitochondria at equivalent values of A$. Thus the “saturation point” is reached at lower A$ values when the dye/protein ratio is low. A similar reasoning explains also why at constant dye/protein ratios, the lower the dye concentration, the higher is the value of A$ at which the deviation from linearity initiates. In conclusion our data indicate that the linearity of the safranine response increases with the increase of dye/ protein ratios. However at high dye/protein ratios calibration of the safranine response with I(+ diffusion potentials is hindered by the amount of loss of matrix K+ in exchange with safranine. A third problem is the time response of the dye. As shown in Fig. 7 the relationship between tliZ and amounts of protein or of dye is hyperbolical. This is not unexpected for a probe monitoring,A$ by redistributing across the membrane since this renders the t ,,p dependent on the rate and amount of dye transport across the membrane. The tlr2 of the dye response remains in the range of 3-5 s below a dye/protein ratio of 12. At higher dye/protein ratios the t1,2 increases considerably, and is higher than 20 s above a dye/protein ratio of 50. There is an inverse correlation between conditions for linearity of dye response and for decrease oftliZ, since the faster probe response occurs at the low dye/protein ratios where there is a more marked deviation from linearity. This represents a limit to the utilization of the dye.
264
ZANOTTI
AND AZZONE
A fourth problem is the toxic effects of the dye, expected for any extrinsic probe above certain concentrations. Figure 8 shows that the uptake of safranine leads to inhibition of electron transport, of energy coupling, and of energy transfer. However these effects occur at different dye concentrations. The effect on energy coupling is negligible since, at a dyeiprotein ratio of 80, under conditions where almost all the dye is inside the mitochondria and bound to the membrane, the stimulation of the state 4 respiration is about 10%. More marked is the effect on the respiratory rate since 50% inhibition of the FCCP-stimulated respiration occurs at a dye/protein ratio of 150, but under conditions where the dye uptake is negligible. The more marked effect of the dye appears to consist in abolishing the ADP-induced stimulation of respiration. Both the inhibition of e- transfer and of ADP induced respiration are consistent with the view that the dye, a lipophilic cation, acts as an inhibitor of the H+ channels, both at the level of the redox complex and of the ATPase. Effects similar to those observed here with safranine have been reported for other extrinsic probes acting as permeant lipophilic cations and thus presumably taken up, accumulated and bound to the membrane (8, 30, 31). This suggests that the toxic effects of safranine are nonspecific. The experiment of Fig. 10 is the first attempt to determine the amount of oxygen uptake required to charge the mitochondrial membrane, during succinate oxidation, at the level of the static head potential. The dimension of the electrical field is followed on the basis of the rate of safranine uptake. We show in Fig. 9 that this rate increases linearly with AI/J. Hence the maximal rate of safranine uptake corresponds to the maximal AJI existing in mitochondria which is, by definition, that found under static head conditions. The minimal amount of oxygen to reach the maximal rate of safranine uptake then becomesthe amount of respiration required to induce a metabolic transition from anaerobiosis to energization, i.e., the extent of charge separation, due to the redox H+ pump, to increase A$ from zero to the static head level. This amount of oxygen is about 0.5 natom x mg prot-’ correspond-
ing, on the basis of stoichiometry of 8 H+/O, to the extrusion of 4 nmol H+ x mg prot-‘. This value is in reasonable agreement with the calculation made by Mitchell (29) of 1 nmol H+ x mg prot- I. The agreement between experiment and prediction indicates that the two values on which the prediction was based are presumably correct: (i) a mitochondrial surface of 40 m’lg prot; (ii) a capacitance of the mitochondrial membrane of 1 pF/cm2. The low amount of oxygen uptake required to raise A$ to the steady state level explains the requirement for counter ions for any microscopic ion flux in mitochondria. This requirement permits the replacement of H+ with cations for any H+ pump stoichiometry measured in steady state, i.e., involving an amount of oxygen uptake greater than 0.2 natom oxygen x mg prot-’ . The above considerations however should not underestimate a fundamental discrepancy. While 0.5 natom x oxygen x mg prot-’ is required to charge the membrane, protons are extruded in the presence but not in the absence of permeant cations (3234). Why protons are not seen after oxygen pulses in the absence of permeant cations is obscure. A reasonable explanation is that the basic event in energy transduction is the formation of an electrical field within the membrane. This is followed by H+ extrusion in the outer aqueous phase only when permeant cations move across the membrane down the electrical field. REFERENCES 1. ROTTENBERG, H. (1975) J. Bioenerg. 7, 61-74. 2. AZZONE, G. F., BRAGADIN, M., POZZAN, T., AND DELL’ANTONE, P. (1976) Biochim. Biophys. Acta 459, 96- 109. 3. AZZI, A., GHERARDINI, P., AND SANTATO, M. (1971) J. Biol. Chem. 246,2035-2042. 4. JASAITIS, A. A., KULIENE, V. V., AND SKULACHEV, V. P. (19’71) Biochim. Biophys. Acta 234, 177- 181. 5. RADDA, G. K., AND VANDERKCMX, J. (1972) Biochim. Biophys. Acta 265, 509-549. 6. FERGUSON, S. J., LLOYD, W. S., AND RADDA, G. K. (1976) Btichim. Biophys. Acta 423, 174- 188. 7. LARIS, P. C., BAHR, P. D., ANDCHAFFEE, R. R. J. (1975) Biochim. Biophys. Acta 376,415- 425.
SAFRANINE
AS MEMBRANE
8. KINNALLY, W. K., TEDESCHI, H., AND MALOFF, B. L. (1978) Biochemistry 17,3419-3428. 9. KINNALLY, W. K., AND TEDESCHI, H. (1976) FEBS Lett. 62, 41-45. 10. AKERMAN, K. E. O., AND WIKSTROM, M. F. (1976) FEBS Lett. 68, 191-197. 11. COLONNA, R., MASSARI, S., AND AZZONE, G. F. (1973) Eur. J. Biochem. 34, 577-585. 12. AKERMAN, K. E. O., AND SARIS, N. E. L. (1976) Biochim. Biophys. Acta 426,624-629. 13. COHEN, L. B., AND SALZBERG, B. M. (1978) Rev. Physiol. Biochem. Pharrnacol. 83, 36-88. 14. ZANOTTI, A., AND AZZONE, G. F. (1979) in XIth lnternational Congress of Biochemistry, Toronto, Canada. 15. MASSARI, S., BALBONI, E., AND AZZONE, G. F. (1972) Biochim. Biophys. Acta 283, 16-22. 16. NICHOLLS, D. G. (1974) Eur. J. Biochem. 50, 305-315. 17. AZZONE, G. F., POZZAN, T., MASSARI, S., AND BRAGADIN, M. (1978) Biochim. Biophys. Acta 501, 296-306. 18. AZZONE, G. F., POZZAN, T., AND MASSARI, S. (1978). Biochcm. Biophys. Acta 501, 307-316. 19. ROSSI, E., AND AZZONE, G. F. (1969) Eur. J. Biochem. 7, 418-426. 19a. BRAGADIN, M., POZZAN, M., ANDAZZONE, G. F. (1979) Biochemistry 18, 5972-5978. 20. PAL, M. K., AND SCHUBERT, M. (1962) J. Amer. Cherrr. Sot. 84. 4384-4393.
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PROBE
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21. WAGGONER, A. S. (1976) J. Membrane Biol. 27, 317-334. 22. STONE, A. L., AND BRADLEY, D. F. (1961) J. Amer. Chem. Sot. 83, 3627-3634. 23. STONE, A. L., AND BRADLEY, D. F. (1967) Biochim. Biophys. Acta 148, 172-192. 24. VITAGLIANO, V., AND COSTANTINO, L. (1970) J. Phys. Chem. 74, 197-202. 25. HLADKY, S. B., AND RINK, T. J. (1976)J. Physiol. 263, 287-319. 26. HOFFMANN, J. F., AND LARIS, P. C. (1974) J. Physiol. 239, 519-552. 27. KETTERER, B., NEUMCKE, B., AND LAOGER, P. (1971) J. Membrane Biol. 5, 225-245. 28. TSIEN, R. Y., AND HLADKY, S. B. (1978)d. Merrcbrane Biol. 38, 73-97. 29. MITCHELL, P. (1966) Biol. Rev. 41,445-502. 30. HIGUTI, T., YOKOTA, M., ARAKAKI, N., HATTORI, A., AND TANI, I. (1978) Biochfim. Biophys. Acta 503, 211-222. 31. GRIMWOOD, B. G., AND WAGNER, R. P. (197(i) Arch. Biochem. Biophys. 176, 43-52. 32. CHAPPELL, B. J., AND HAARHOFF, K. (1967) i?l The Biochemistry of Mitochondria (Slater, E. C., et al., eds.), pp. 75-91, Academic Press, New York/London. 33. CHANCE, B., AND MELA, L. (1966) Nature (LOW don) 212, 372-376, 34. POZZAN, T., AND AZZONE, G. F. (1976) FEBS Lett. 71, 62-66.