Eur. J. Biochem.

50, 305-315 (1974)

The Influence of Respiration and ATP Hydrolysis

on the Proton-Electrochemical Gradient across the Inner Membrane
of Rat-Liver Mitochondria as Determined by Ion Distribution
David G. NICHOLLS
Wenner-Grens Institut, Stockholms Universitet
(Received May 22iAugust 28, 1974)

1. A technique is described, based on the distribution of rubidium, acetate and methylammonium

ions, for the simultaneous estimation of membrane potential and pH gradient across the inner
membrane of mitochondria. The technique requires less than 0.5 mg mitochondrial protein and is
independent of many factors which interfere with electrode determinations of protonmotive force
@PI.
2. With a limiting matrix volume of 0.4 pl/mg mitochondrial protein, the indicated value of Ap
for rat liver mitochondria is 228 mV in state 4, 170 mV in state 3, and -0.6 mV in the presence of
rotenone and uncoupler. The relative contributions of the pH gradient and membrane potential are
dependent on the availability of electrophoretically and electroneutrally translocatable species in the
incubation medium.
3. In a sucrose-based medium containing 0.5 mM KCl, rotenone and uncoupler, the technique
indicated a membrane potential of + 85 mV and a pH gradient of + 1.46 (acidic in the matrix
compartment). In state 4, under no conditions examined did the pH gradient contribute more than
50 % of the total protonmotive force.
4.The hydrolysis of ATP generates an optimal Ap of 220 mV.
5 . The proton conductance of the inner membrane is potential dependent, increasing when Ap
is greater than 200 mV.
6. The extra-mitochondria1 phosphate potential sustainable by respiration was found to change
in parallel to Ap, but to exceed the latter parameter when based upon a stoichiometry of two protons
translocated per ATP synthesised.
The determination of the electrochemicalpotential
gradient of protons (dpH+) across the inner membrane
of mitochondria is central to the experimental verification of the chemiosmotic theory of energy transduction [l - 31. Two broad criteria must be satisfied,
firstly that qualitative changes in the magnitude of
the gradient should occur under conditions predicted
by the theory, and secondly that the gradient should
be quantitatively sufficient at all times to account for
the observable equilibrium phosphate potential sustainable by respiring mitochondria.
In contrast to the extensive literature on the
magnitude of the phosphate potential during controlled respiration [4- 121 there have been very few
attempts to confirm or extend the original deterAbbreviation. FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone.
Eur. J. Biochem. 50 (1974)

minations by Mitchell and Moyle [3] of the magnitude

of the protonmotive force (Ap, equal to ApH+/F,
where F is the Faraday). Recent reports [13,14] have
determined values of Ap which differ from those
obtained by Mitchell and Moyle [3] both qualitatively
and quantitatively.
In order to attempt to resolve this controversy,
the present communication describes a technique for
the simultaneous determination of the electrical and
chemical components of Ap which is free from many
of the experimental restrictions inherent in the ionspecific electrode technique of Mitchell and Moyle [3]
and additionally is sufficiently sensitive to enable
multiple analyses to be performed on small quantities
of mitochondria.
The value of Ap obtained during state 4 respiration
is in full agreement with the original estimation [3].
In addition, Ap was estimated during state 3 respira-

Mitochondria1 Proton-Electrochemical Gradient

306

tion and during ATP hydrolysis and it was possible

to confirm that protons were in electrochemical
equilibrium across the inner membrane in the absence
of respiration and in the presence of uncoupler.
The apparent proton conductance of the inner
membrane, which was found by Mitchell and Moyle
[15] to be potential-independent under anaerobic
conditions, is found in the present study to increase
when Ap is in excess of 200mV, suggesting that the
value of 230mV obtained for Ap during state4
respiration might be determined by the electrical
properties of the inner membrane rather than by
thermodynamic equilibrium with the respiratory chain.
Perhaps the most serious objection to the current
formulation of the chemiosmotic theory is that the
magnitude of the protonmotive force is inadequate
to account for the observed phosphate potential [412,161. In order to remove differences in experimental
technique as a possible contributary factor to this
discrepancy, the phosphate potential and protonmotive force were determined under conditions which
were designed to be closely parallel. Employing the
value for the standard free energy of ATP hydrolysis
determined by Rosing and Slater [9], the two parameters were found to vary in parallel, but the phosphate
potential was found to be 50 to 80 mV in excess of Ap.

MATERIALS AND METHODS

Mitochondria were prepared from rat liver by
differential centrifugation in 0.25 M sucrose. Oxygen
uptake was determined by a Clark-type oxygen
electrode in a perspex chamber of 0.9 ml capacity.
All experiments were performed at 23"C, and the
initial pH of the incubation media was 7.2. Protein
was determined by the biuret method.
Oligomycin, rotenone, sodium succinate, sodium
~~-3-hydroxybutyrate,
ATP and ADP were obtained
from Sigma Chemical Co. ; valinomycin from Calbiochem, and all isotopes from the Radiochemical Centre
(Amersham, England). FCCP was a gift from Dr P. G.
Heytler. All other reagents were of the highest purity
commercially available.
The Determination of Protonmotive Force

Protonmotive force (Ap), as defined by Mitchell

and Moyle is related to the components of ApH' at
23 "C thus
Ap = APH'/F = A E - 59ApH.

(1)
Where F is the Faraday, Ap the protonmotive force in
mV, AE the membrane potential in mV and ApH the
pH gradient across the inner membrane [l - 31.

The membrane potential component of Ap is here

determined from the distribution of 86Rb+across the
inner membrane in the presence of valinomycin, with
the assumption that the cation distributes according
to a Donnan equilibrium [3].

where [M+Iiand [M'l0 represent the activities of the

cation in the internal (matrix) and external (medium)
phases, respectively. Control experiments demonstrated that the electroneutral cation/proton exchange
activity of the inner membrane [I71 transported Rb'
very slowly, thus diminishing disturbance of the "Rb+
gradient by dpH.
While the distribution of a weak acid across the
inner membrane has been frequently employed to
estimate ApH [14,16,18- 201, technical reasons prevent accurate determinations when ApH is positive
(matrix compartment more acidic), a condition reported by Mitchell and Moyle [3] for the de-energised
mitochondrion. In order to provide an accurate
determination of ApH under all conditions, a weak
base and a weak acid are present simultaneously in
the incubation medium.
The ability of a weak acid or base to monitor ApH
is based on the assumption that the concentration
(strictly the activity) of the respective uncharged forms
(HA and B, respectively) are equal on both sides of
the membrane. If the membrane is impermeable to
the charged species (A- and BH+), the distribution
of the various species will be governed by the Henderson-Hasselbach equation, and if the pK, of the acid
and base are sufficiently removed from the pH of the
incubation, then the following relationship may be
derived [19,20]
(3)
Where i and o represent the matrix and extra-matrix
compartments respectively.
In the present communication, acetate (pK, 4.72)
and methylammonium (pK, 10.62) are employed as
the weak acid and base respectively. The suitability
of these indicators will be discussed below.
The Practical Determination ?f AE and ApH

In order to determine the components of the

protonmotive force in respiring mitochondria by ion
distribution it is essential that the mitochondria are
separated from the medium by a method which avoids
anaerobiosis. The decay of Ap on interrupting the
source of energy is sufficiently rapid (Fig. 7) to cause
Eur. J. Biochem. 50 (1974)

D. G. Nicholls

substantial loss of the ion gradients if the separation

procedure requires more than a few seconds. Simple
centrifugation procedures result in inevitable anaerobiosis in the pellet. Thus a 2-min centrifugation
and concurrent anaerobiosis would be expected to
lead to a decrease of at least 50 mV in A p before
samples were analysed (Fig. 7), and may explain some
discrepancies between the maximal values of Ap
and those obtained by different workers [3,14]. This
problem is not encountered during rapid membranefilter filtration, as fresh incubation medium continuously filters through the film of mitochondria.
In order to calculate the degree of concentration
of the isotopic indicators within the mitochondrial
matrix, the matrix volume and the volume of incubation medium, including the inter-membrane space,
entrapped in the membrane filter, must be accurately
determined. The matrix volume may be calculated as
the sucrose-impermeable, water-permeable space, by
centrifugation of non-respiring mitochondria. Membrane filtration is not sufficiently accurate for this
determination, due to the high volume of the entrapped medium on the filter. It is not therefore
possible to take account of respiration-dependent
swelling of the mitochondrion, and the matrix volume
may be underestimated, and hence the components of
dp over-estimated, under conditions where respirationdependent swelling is extensive (see Fig. 3). The
approach adopted in this communication is to employ
the limiting value for the matrix water (0.4 pl/mg
mitochondrial protein [21]) except under conditions
where uptake of potassium (calculated from the
rubidium distribution and the external potassium concentration) is sufficient to cause appreciable swelling
of the matrix; in which case the matrix volume is
calculated by assuming osmotic equilibrium, with the
matrix potassium contributing 50 % of the internal
osmolarity [22]. In practice, this correction is only
required for mitochondria respiring in state 4 when
the initial external potassium concentration is in
excess of 1 mequiv./l.
For filtration, Sartorius 0.6 pm cellulose nitrate
membrane filters are employed. The filter support is
the male half of a 13 mm pressure filter holder. Under
vacuum the membrane filter seals onto the holder,
forming a depression which can hold 0.5 ml of incubation. Filtration is normally complete in under 5 s.
Incubation media are prepared containing all three
indicators, [3H]acetate, [4C]methylammonium and
Rb. 3H, 14C and 86Rb have maximum particle
energies which are sufficiently separated (0.018, 0.156
and 1.7 MeV, respectively) to enable the three isotopes
to be counted simultaneously with good separation
on a 3-channel liquid scintillation counter. [3H]Acetate
(10 pM, 0.5 pCi/ml), [14C]methylamine (10 pM,
Eur. J. Biochem. 50 (1974)

307

0.1 pCi/ml) and RbC1 (10 pM, 0.05 pCi/ml) are

present in the basic incubation mixture, which also
contains 250 mM sucrose, 10 mM choline chloride,
10 mM glycyl-glycine pH 7.2, and 0.5 pM valinomycin.
After making any further additions, mitochondria
(1 mg protein/ml) are added and 2 to 4 min allowed
for equilibration. 400 pl aliquots are then taken and
filtered through the membrane filters. The filters are
then added directly, without washing, to scintillation
vials containing Triton X-100 2 ml, toluene 4 ml,
H 2 0 0.6 ml and 2,5-diphenyloxazole5 g/l. In addition,
15 pl aliquots of the basic incubation mixture are
spotted onto membrane filters, which are then added
to scintillation vials as above. In this way the filtered
mitochondria and the incubation mixture can be
counted under conditions of quenching which are as
closely similar as possible. On shaking for a few hours,
both the mitochondria and the membrane filters dissolve in the scintillant to give a clear homogeneous
solution which may then be counted to determine
the activity of the three isotopes.
Under the conditions employed, the membrane
filter absorbs about 15 pl medium, and the counts
attributable to this extra-matrix volume must be
carefully allowed for before the counts within the
matrix can be calculated. The extra-matrix volume
is not calculated directly (for example by determination
of the sucrose-permeable space), but instead use is
made of the prediction from Eqns ( 5 ) and (6), that the
acetate and methylammonium distributions vary inversely with dpH, acetate being excluded from the
matrix when dpH is positive, and methylammonium
being excluded when ApH is negative, enabling these
ions to be used for the calculation of extra-matrix
volume under the appropriate conditions.
The total counts attributable to each isotope in
the aliquot to be filtered is first calculated from the
controls taken from the incubation before addition
of mitochondria. Next the apparent space indicated
by each isotope on the membrane filter containing
the film of filtered mitochondria is calculated as though
the isotope had retained the same concentration on
the filter as in the filtrate from that sample,
Space

V
N

~- 1

Pl

(7)

n
where V is the volume of filtrate, N the total counts
of the isotope in the aliquot before filtration and n
the counts of the isotope retained on the membrane
filter. If these spaces are r, c and h for rubidium,
methylammonium and acetate, respectively, and the
matrix volume, determined as the sucrose-impermeable space, is m,then the the degree of concentration

Mitochondria1 Proton-ElectrochemicalGradient

308

(]I:

0.1

4'
0

1 .o

0.5

1.5

l3 PH

Fig. 1. ApH calculatedjrom Eqn (9) as a junction of the matrix

volume (m) and the apparent 'spaces' associated with the
membranefilter plus mitochondria for [14C]methylammonium
(c) and [3Hjocetute (h), respectively. The relation between
- A p H and (h - c)/m is identical

which closely approximated to those for the determination of A p . The incubation medium contained
250 mM sucrose, 10 mM choline chloride, 10 mM
glycyl glycine, 0.5 pM valinomycin, 0.46 mM KCI,
0.1 mM sodium-ADP, 1 pCi/ml [3H]ADP, 1 mg/ml
albumin, 0.93 mM sodium phosphate and 1 mM
sodium-EDTA. Further additions are detailed in the
text. Mitochondria, 1.3 mg/ml, were added to the
incubation, and 3 min allowed for equilibration of
the added [3H]ADPwith the matrix adenine nucleotide
pool. 5 mM sodium 3-hydroxybutyrate was then
added, and a further 4min allowed for equilibrium
to be attained. The incubation was filtered through
a 0.6 pm cellulose nitrate membrane filter, and the
filtrate was quenched in PCA. Neutralised extracts
were separated on polyethylimine cellulose thin-layer
plates, eluting with 1 M LiCI. Spots were eluted with
10 mM HCL, 1 M NaCl, and counted for tritium.

RESULTS AND DISCUSSION

of each isotope within the matrix is given by the
following relationships :

where u is the extra-matrix volume of incubation

entrapped in an individual filter.
Combining Eqns (9,(6), (8b) and (8c):
C - v

h-v
m

C - h
m

C - h
The dependency of
upon ApH calculated from
Eqn (9) is plotted in Fig. 1. Once ApH is known, the
exact value of u can be determined from Eqns (5), (6)
and (8), to enable the degree of concentration of
rubidium to be determined.

However, in practice, negligible errors are introduced

if v is set equal to h when dpH > 0 and equal to c
when ApH < 0.
The extent of phosphorylation of extra-mitochondrial [3H]ADP was determined under conditions

Rat liver mitochondria incubated in the basic

medium, with the addition of 0.5 mM KCI, the indicators [3H]acetate, ['4C]methylammonium and
86Rb+,and 3-hydroxybutyrate as substrate, accumulate acetate and rubidium within the matrix (Table 1B).
The apparent acetate and rubidium 'space' within
the matrix represent a 26-fold accumulation of acetate
and a 340-fold accumulation of rubidium, assuming
a matrix volume of 0.4 pl/mg mitochondrial protein [3]. These concentration gradients indicate a
membrane potential, A, of 150 mV [Eqn (2)] and a
pH gradient, ApH, of - 1.41 [Eqn (3)]. The protonmotive force, A p , calculated from Eqn (1) is 227.6
f 7.1 mV (mean of 18 experiments).This value should
be compared with 223 f 5 mV obtained by Mitchell
and Moyle [3] under conditions which the experiment
in Table 1 was designed to imitate as closely as possible. The concentration gradients of acetate and
rubidium across the mitochondrial membrane attain
a constant value within 60 s of the initiation of the
incubation, and are thereafter constant for at least
5 min. Routinely 2 min is allowed for equilibration
of the indicators. The apparent spaces within the
matrix under these conditions are directly proportional
to the quantity of mitochondrial protein entrapped
upon the membrane filter (Fig. 2).
The triple-label technique developed in this paper
will only be valid if the extra-matrix volume is given
exactly by the acetate space when ApH > 0 or by the
methylammonium space when ApH < 0 (see Methods).
This was confirmed by control experiments under the
conditions of Table 1, except that each isotopic indicator was present in a separate incubation, the extraEur. J. Biochem. 50 (1974)

D. G. Nicholls

309

Table 1. The calculation of Ap, AE and ApH,for rat liver mitochondria

Rat liver mitochondria (1 mg protein/ml) were incubated in
the basic medium (see Methods), with the further additions
in Expt A of 0.5 mM KC1; 5 pM rotenone and 1 p,M FCCP,
or, in Expt B, with the additions of 0.5 mM KCl and 5 mM
sodium 3-hydroxybutyrate. Aliquots were filtered and counted
as described in Methods. All counts are corrected for crossover and background. The matrix volume was taken to be
0.4 &mg mitochondria1 protein
Radioactivity in 400 1

Calculation for

counts/min
[jH]Acetate
['4C]Methylammonium
86Rb+

268 133
71 306
11200
Membrane filters
A

Mitochondria1 protein (mg /assay)

counts/min
-

11638
3 844
61 1

13501
2912
1712

Apparent 'spaces'
on membrane filters

18.15
22.79
23.08

h ([3H]acetate)
c (['4C]methylammonium)
r ("Rb')

21.21
17.03
72.17

Concentration in matrix
[Eqn @)I
A
[Aclii [Aclo
[CH,NH21 i/[CHSNHz lo
[Rbl i/[Rblo
- 59 ApH (from Fig. I)
AE [from Eqn (2)]
Ap [from Eqn (I)]

B
26. I
29.0
30.1
- 86

+ 88
+ 2

344.4

+ 83
+ 150
+ 233

matrix volume being determined by ['4C]sucrose or

[3H]sucrose.In the presence of substrate, when ApH
= - 1.41 (Table 1) the ['4C]methylammonium and
[3H]sucrose spaces on the membrane filter in the
presence of 0.5 mg mitochondrial protein were respectively 11.22 p1 and 11.59 p1 (means of 9 determinations). Similarly, in the presence of rotenone and
FCCP when ApH is 1.46, the [jH]acetate and [I4H]sucrose spaces were 16.26 pl and 16.37 pl respectively.
Thus it is justified to use acetate and methylammonium as indicators of the extra-matrix volume

Eur. J. Biochem. 50 (1974)

Fig. 2. The apparent acetate and rubidium 'spaces' within the

ma1ri.x of rat liver mitochondria oxidising 3-hydroxybutyrate,
as a function of mitochondrialprotein entrapped upon thefilter.
Conditions were identical with those in Table 1 B, except
that the mitochondrial protein was varied. The initial external
KCl concentration was 0.25 mM. (0) apparent rubidium
'space', (0)apparent acetate 'space'

under conditions when Eqns (5) and (6) predict an

exclusion from the matrix. These results also eliminate
the possibility of significant error induced by binding
of these indicators to the mitochondria [20], or of
acetate accumulation as a consequence of an anionexchange mechanism with anions initially present in
the matrix [23], as neither of these effects would be
sensitive a priuri to the transmembrane pH gradient.
Furthermore, the failure to detect an apparent methylammonium-permeable sucrose-impermeable space in
the presence of rotenone and FCCP eliminates methylammonium accumulation by lysosomal contamination
of the mitochondrial fraction as a source of serious
error, as such accumulation would, of course, be
insensitive to changes in the energisation of the
mitochondria.
The relative magnitude of the membrane potential
(AE) and pH gradient (- 59 ApH) components of the
protonmotive force is dependent on the extent to
which electrophoretic cation translocation can occur [3]. The components of Ap as a function of the
initial external potassium concentration are plotted
in Fig.3A for mitochondria in the presence of
3-hydroxybutyrate, and assuming a constant matrix
volume of 0.4 pl/mg mitochondrial protein. The
apparent protonmotive force is constant while the
magnitude of - 59 ApH and AE approach each other
assymptotically tending towards an equipartition of
potential between the two components. Under no

Mitochondria1 Proton-ElectrochemicalGradient

310
250

250

200

;
150
E

100
50
0
250

t
200

150

04
0

3 1oc

6
[Phosphate] ( m M )

10

5c

Fig. 4. Ap as a function of the initial external phosphate concentration for rat liver mitochondria oxidising 3-hydroxybutyrate. Conditions were as for Table 1 B, with the further

addition of the indicatedconcentrationsof sodium phosphate.

(A) Ap, (0)AE, (0)- 59ApH
0

4
6
[KCII (mM)

10

Fig. 3. Ap as a function of the initial external potassium concentration for rat liver mitochondria oxidising 3-hydroxybutyrate. Conditions were as for Table 1 B, except that the
initial external potassium concentration was varied. (A) Assuming a constant matrix volume of 0.4 pl/mg; (B) assuming
osmotic equilibrium with potassium contributing 50 % of the
matrix osmolarity. (A) Ap, (0)AE, (0) - 59 ApH

circumstances was the pH component of Ap observed

to be greater than the AE component. This is in contrast to the results reported by Mitchell and Moyle [3]
where, in the presence of 10 mequiv./l of potassium,
the pH component greatly exceeded AE in magnitude.
It is clear from Fig.3A that the assumption of a
constant matrix volume does not hold under conditions where a considerable accumulation of cation
occurs. If the matrix compartment behaves as a perfect
osmometer [24] and potassium ions are assumed to
contribute 50% of the osmotic support of the matrix
[22] and to distribute as rubidium, then the matrix
volume can be calculated. Thus when the external
potassium concentration is 6 mequiv./l, a matrix
volume of 1.47 pl/mg mitochondrial protein is required
for osmotic equilibrium. The potentials plotted in
Fig. 3B are corrected for this expansion of the matrix
volume. It should be noted that errors in matrix
volume do not affect the relative contributions of the
pH gradient and AE. In subsequent experiments, the
potassium concentration of the medium was kept
below 1 mequiv./l, and the limiting value for the

matrix water of 0.4 pl/mg mitochondrial protein [3,21]

was assumed.
Just as an increase in the concentration of an
electrophoretically translocatable ion leads to a decrease in AE, an increase in an electroneutrally translocatable species such as phosphate [24] leads to a
decrease in ApH (Fig.4). It can be seen that under
these conditions (in the presence of valinomycin) Ap
falls rapidly as the external phosphate concentration
is increased.
The fall in the steady-state protonmotive force on
increasing the concentrations of potassium or phosphate under the conditions of the experimentsdepicted
in Fig. 3 and 4 should, if these values accurately reflect
the energy level of the mitochondrion, be associated
with a corresponding fall in the equilibrium phosphate
potential which these mitochondria can maintain.
In Table 2, phosphate potential [4- 121 is expressed
in terms of the millivolts of protonmotive force required to maintain the equilibrium on the assumption
that two protons are translocated into the matrix
compartment for every molecule of ATP appearing
in the extra-mitochondria1 compartment. It may be
seen that while the phosphate potential and protonmotive force change in parallel, the former parameter
indicates a requirement for a potential some 50 - 80mV
in excess of that recorded as the protonmotive force.
The chemiosmotic theory predicts a protonmotive
force of zero in the absence of respiration and in the
presence of an uncoupler, and this was assumed in
the original determinations of Ap [3]. The isotope
Eur. J. Biochem. 50 (1974)

D. G . Nicholls

31 1

Table 2. The relation between the extra-mitochondrial phosphate potential and protonmotive force j o r rat liver mitochondria
oxidising 3-hydroxybuzyrare
The experiment was performed as described in Methods. Further additions to the incubation medium are detailed below. The
extra-mitochondria1ATP/ADP ratio was determined 4 min after the initiation of respirationby the addition of 3-hydroxybutyrate.
Phosphate potential was varied by the addition of varying concentrations of potassium or phosphate to the medium. The
phosphate potential was calculated on the assumption of a stoichiometry of two protons translocated per ATP synthesised,
employing a value of - 7.9 kcal/mol for the standard free energy of hydrolysis of ATP [9]. Values for Ap were obtained from the
experiments depicted in Fig. 3 and 4

Kf

Pi

ADP

ATP/ADP

FM

mM

__

0.46
0.91
1.91
2.91
4.91
0.46
0.46
0.46
0.46
0.46

ATP

0.93
0.93
0.93
0.93
0.93
1.43
2.43
3.43
5.43
7.93

82
79
47
17
5
84
87
87
88
89

7
16
48
77
88
6
6
5
5
5

distribution technique enables this prediction to be

tested experimentally. In 19 experiments the mean
value of Ap for rat liver mitochondria in the presence
of rotenone and FCCP was - 0.6 mV. However the
individual values of dpH and A E were considerably
larger than those reported by Mitchell and Moyle [3],
the mean values of - 59 ApH and AEbeing respectively
- 86 mV and
85 mV in the presence of 0.5 mM
KCl (Table 1B) in contrast to - 33 mV and 33 mV,
the values obtained by the previous authors. It is
unlikely that these values are artifactually high due
to an underestimation of the matrix volume, as rat
liver mitochondria assume a highly condensed matrix
conformation in a sucrose medium in the presence of
uncoupler and valinomycin [21]. Identical values of
AE are obtained in dual-label experiments where the
extra-matrix volume is determined with ['4C]sucrose.
A protonmotive force of zero is predicted from
thermodynamic principles under these conditions, thus
this result in turn supports the validity of the ion
distribution technique, and suggests that errors due
to binding of indicators to the mitochondrion, or due
to an undefined activity coefficient within the matrix,
do not greatly distort the isotope distribution.
The simplest explanation for the origin of the pH
gradient and membrane potential in this state would
be in terms of a Donnan effect, the impermeant
anions within the matrix functioning as a cation
exchanger, the tendency for potassium to flow out
of the matrix down its concentration gradient, catalysed by valinomycin, being countered by the entry

Eur. J. Biochem. 50 (1974)

11.4
5.1
1.0
0.22
0.055
13.1
17.8
17.2
16.9
19.2

[ATPI
[ADP] [Pi]

Phosphate
potential

M-'

mV

13 400
5 990
1100
240
59
9 700
7 600
5 100
3 200
2 400

292
282
261
241
223
288
285
280
274
27 1

Ap

230
214
1 94
186
175
210
204
200
191
184

of protons, catalysed by FCCP, in response to the

generated membrane potential. Thus, when the membrane potential is decreased by increasing the external
potassium concentration (Fig. 5), there is a parallel
decrease in ApH, so that Ap remains at zero. In the
presence of 1 mM external KC1, the matrix potassium
content, calculated from AE and the matrix water,
and assuming an activity coefficient of unity, is
30 nequiv./mg mitochondria1 protein, a figure comparable to previously published results [22].
The determination of Ap during coupled oxidative
phosphorylation by employing ion-specific electrodes
[3] is complicated by proton uptake accompanying
the phosphorylation of ADP. This interference is
absent in the present technique, and Fig.6 demonstrates the changes in the components of Ap for
mitochondria oxidising 3-hydroxybutyrate upon addition of 0.3 mM ADP. There is an immediate and
rapid fall in dp from 220 mV to 170 mV during the
transition from state 4 to state 3. As the ADP is
phosphorylated, the protonmotive force increases
steadily, and when the cycle of state 3 respiration is
completed, returns to its initial value of 220 mV.
These results suggest that there is a measure of equilibration between the phosphate potential and the
protonmotive force, as the two increase in parallel
during the cycle. It should be stressed that as these are
not steady-state conditions, the phosphate potential
and protonmotive force will not be in true equilibrium.
However it may be noted that in the middle of the
phosphorylation cycle, when ATP/ADP in the medium

Mitochondria1 Proton-Electrochemical Gradient

312
250
100

200

50

>
-E

->

150

-E
4

1oc

-5c

-1oc

5c

C
10

20

30
40
[KCII (mM)

50

60

70

Fig. 5. The components of Ap for rat liver mitochondria in the

presence of rotenone and FCCP as a function of the initial
external potassium concentration. Concentrations were as for
Table 1A, except that the KCI concentration was varied as
indicated, the sucrose concentration being decreased to
maintain the same nominal osmolarity. (A) Ap, (0) AE,
(0)- 59ApH

Fig. 6. The componentsofAp,for rat liver mitochondria oxidising

3-hydroxybutyrate during u cycle of ADP phosphorylation.
Conditions were for Table 1B with the further additions of
2 mM phosphate and 2 mM MgCI,. At the point indicated
by the arrow 0.3 mM ADP was added. (A) Ap, (0) AE,
(0)- 59dpH
250

will be close to unity, Ap, in the presence of 2 mM

phosphate, is 181 mV. These values are in rather
good agreement with those originally estimated by
Mitchell and Moyle [3] but diverge widely from those
recently published by Padan and Rottenberg [I41 who
obtained 148mV and 143mV in states 4 and 3
respectively.
The chemiosmotic theory requires that a protonmotive force may be generated either by respiration
or by ATP-hydrolysis. This is demonstrated in Fig. 7.
Mitochondria were incubated in a medium containing
rotenone. It may be noted that the protonmotive force
is not immediately zero, as is the case if FCCP is
present (Fig. 5). On addition of ATP there is a very
rapid energisation of the mitochondria, and a Ap of
220 mV is obtained, a value identical to that obtained
under state 4 respiration. While there is a relatively
small increase in AE, from 97 mV to 126 mV, the pH
gradient changes from + 1.21 to - 1.55, increasing
the contribution of the pH gradient to Ap by 160 mV.
On addition of oligomycin there is a slow decay of
Ap with a half-time of 140 s, which is compatible with
the half-time for proton equilibration across the inner
membrane [25].

4
Time ( m i n )

J.

m
150

-> 100
E

50

c
-5c

-100

4
6
Time (rnin)

10

Fig. I. The components ofdp for rat liver mitochondria during

the hydrolysis of ATP. Mitochondria (1 mg protein/ml) were
incubated in the basic medium with the additions of 0.5 mM
KCI; 5 pM rotenone and 2 mM MgC12.At the time indicated
by the first arrow, 1 mM ATP was added. The second arrow
represents the addition of 1 lg/ml oligomycin. (A) dp,
(0)AE, (0)- 59 ApH
Eur. J. Biochem. 50 (1974)

D. G. Nicholls

250

313

10
20
30
Proton current (nequiv. rnin-
+

40
*

50

rng-1)

Fig. 8. The steady-state rate ofproton re-entry through the inner

membrane of rat liver mitochondria as a function of Ap.
Mitochondria ( 2 mg protein/ml) were incubated in the basic
medium with the additions of 1 mM KCI; 5 pM rotenone and
4 mM sodium succinate. Incubations were performed in an
oxygen electrode chamber at 23 C. Individual determinations
were performed in the presence of varying sodium malonate
concentrations from 0 to 2.5 mM. After recording respiration
for 2 min, 0.4 p1 aliquots were taken and immediately filtered
to determine the components of LIP.The rate of proton reentry was calculated from the respiratory rate multiplied by
the H+/O ratio for succinate oxidation taken from [21]

by the apparent proton-dielectric strength of the

membrane, rather than by a thermodynamic equilibrium with the energy conservation sites of the
respiratory chain. This could also explain the similar
potential obtained during ATP hydrolysis (Fig. 7).
It should be noted that a potential of 230 mV across
the membrane implies a field strength of at least
250000 V/cm.
Fig.8 also provides an explanation for the longstanding empirical observation that, if the ADP/O
ratio is to be calculated from an oxygen electrode
trace, the best approximation is obtained by assuming
that the state 4 rate of respiration ceases during ADPstimulated respiration [26]. In Fig.6 it was shown
that the mean value of LIP during such a state 3 cycle
was 180 mV. This potential (Fig. 8) is below that at
which apparent dielectric breakdown sets in, and is
thus associated with a considerably lower proton
leakage than occurs in state 4. Similarly, the observation that the ADP/O ratio decreases on approaching state 4 conditions [27] is consistent with
these results as a limitation in the rate of phosphorylation would increase Ap which would in turn disproportionately increase the rate of proton leakage.

CONCLUSION
By the chemiosmotic theory, state 4 respiration is
limited by the proton permeability-of the inner membrane [1,2], a proton circuit being established
composed of a generator (the respiratory chain) and
a resistance (the inner membrane). The apparent
conductance of the inner membrane in terms of
proton re-entry has been estimated in state 4 from
the ratio of the proton current (calculated from the
respiratory rate multiplied by the H + / O ratio), to the
protonmotive force. Mitchell and Moyle obtained a
value of O.ll/nequiv. Hf x min- x mg protein-
x mV- in this way [15]. If the inner membrane
behaves as an ohmic conductor of protons, then there
will be a strict proportionality between the proton
current and Ap. The relationship between proton
current and Ap was investigated in the experiment
depicted in Fig. 8. Mitochondria were incubated with
succinate, the activity of succinate dehydrogenase
being controlled by titration with malonate. The rate
of oxygen uptake was recorded and an aliquot was
filtered to determine Ap. The curve relating proton
current to Ap consists of two regions. Up to 180 mV
the inner membrane behaves as an ohmic conductor
of protons, however at higher potentials an increase
of proton conductance becomes apparent, the proton
current increasing faster than Ap. Thus the figure of
230 mV obtained for Ap in state 4 may well be limited
Eur. J. Biochem. 50 (1974)

The results presented in this communication confirm the value for the protonmotive force obtained
by Mitchell and Moyle [3] for mitochondria during
controlled respiration and extend their observations
by continuously monitoring Ap during state 3 respiration (Fig. 6), and during ATP hydrolysis (Fig. 7). The
absence of a protonmotive force in the absence of
respiration and in the presence of uncoupler, which was
assumed on thermodynamic grounds by Mitchell and
Moyle [3], is here confirmed experimentally (Table 1
and Fig. 5).
Such discrepancies as occur between this communication and the original determination of protonmotive force [3], are limited to conditions where the
latter technique is operating at the limits of sensitivity,
namely in the absence of respiration, and in the
presence of high potassium concentrations.
Thus in the presence of rotenone and FCCP, the
components of the zero protonmotive force measured
by isotope distribution are considerably greater than
the
33 mV and - 33 mV previously reported. A
membrane potential of +33mV corresponds to a
potassium gradient in the presence of valinomycin of
only 3.6-fold [Eqn (2)], which in the absence of added
potassium in the incubation medium, would imply
virtually complete depletion of matrix potassium in
the presence of valinomycin and uncoupler. It may

314

be noted that the determination of membrane potential

in this respiratory state in the original publication was
perforce indirect, being calculated from A pH which
was in turn calculated from the ratio of internal to
external buffering capacity and from the change in
external pH on lysis of the mitochondrion [3]. In
contrast the isotope distribution is relatively direct,
and the observed membrane potential of 85 mV in
the presence of 0.5 mM KCl (Table 1B) corresponds
to a 30-fold accumulation of potassium within the
matrix, or 15 mequiv./l, zero protonmotive force being
maintained by a pH gradient of + 1.4 units. These
potentials are artifactual in the sense that they are
dependent on a low external potassium concentration.
In media containing high potassium concentrations
AE and ApH of non-respiring mitochondria each tend
to zero in the presence of uncoupler (Fig. 5).
The second discrepancy with the original authors
results concerns the relative contributions of A E and
- 59 ApH to the protonmotive force in state 4 in the
presence of increasing concentrations of external
potassium. Mitchell and Moyle obtained, in the
presence of potassium at 10 mequiv./l in the incubation
medium, values of AE and - 59 ApH of 83 mV and
150 mV, respectively, and this has led to the concept
that, when sufficient electrophoretic cation translocation can occur, the membrane potential is discharged [3]. In contrast, in the present results, under
no circumstances was - 59 ApH greater than AE, but
instead, as the concentration of translocatable cation
increased, the contributions of the pH gradient and
the membrane potential to the protonmotive force
tended towards equality (Fig. 3). Thus the pH gradient
never appears to contribute more than one half of
the protonmotive force.
Despite this, the changes in membrane potential
are as a rule very less marked than the changes in
- 59 ApH. Thus, measurements of membrane potential alone [28] can only give a qualitative estimate
of the changes in protonmotive force.
The observation of an apparent increase in the
proton conductance when Ap is greater than 200 mV
(Fig. 8) is based on the assumption that the stoichiometry of proton extrusion by the respiratory chain is
invariant [15]. This assumption is difficult to test
experimentally, as any attempt to determine this
stoichiometry during state 4 respiration would result
in a lowering of Ap. That a protonmotive force of
230 mV represents an upper limit to the dielectric
strength of the inner membrane might be indicated
by the identical protonmotive force generated by the
hydrolysis of ATP (Fig. 7).
While the observation of a transmembrane proton
electrochemical gradient of up to 228 mV which
responds predictably to changes in the energy po-

Mitochondria1Proton-Electrochemical Gradient
tential of the mitochondrion is not evidence per se
that this gradient is the intermediate in ATP production [1,2], failure to observed a gradient with
these characteristics is sufficient to invalidate a chemiosmotic concept of energy transduction.
However, the maintenance of a level of ADP
phosphorylation which requires a free energy in excess
of that which can be donated by the proton electrochemical gradient (Table 2) under comparable conditions is, as has been amply discussed [4- 12,161,
a major obstacle to acceptance of the chemiosmotic
theory in its present form. Unless determinations of
protonmotive force ([3,13,14], this communication)
are substantial underestimates, it is clear that either
a stoichiometry of two protons translocated per ATP
synthesised [29] is not valid under the approach to
equilibrium state 4 conditions, or that the mechanism
of energy transduction is other than chemiosmotic.
This work was supported by a grant from the Swedish
National Science Foundation (2171-025). The author is
grateful to Mrs Agneta Bergstrom for expert technical
assistance.

REFERENCES
1. Mitchell, P. (1966) in Chemiosmotic Coupling in Oxidative
and Photosynthetic Phosphorylation, Glynn Research,

Bodmin, Cornwall.
2. Mitchell, P. (1968) in Chemiosmotic Coupling and Energy
Transduction, Glynn Research, Bodmin, Cornwall.
3. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 7 , 471
-484.
4. Klingenberg, M. (1961) Biochem. Z . 335,243-262.
5 . Cockrell, R. S., Harris, E. J. & Pressman, B. C. (1966)
Biochemistry, 5 , 2326- 2335.
6. Slater, E. C. (1969) in The Energy Level and Metabolic
Control in Mitochondria (Papa, S . , Tager, J. M.,
Quagliarello, E. & Slater, E. C. eds) pp. 255-259,

Adriatica, Bari.
7. Klingenberg, M. (1969) in The Energy Level and Metabolic Control in Mitochondria (Papa, S . , Tager, J. M.,
Quagliarello, E. & Slater, E. C., eds) pp. 189- 193,

Adriatica, Bari.
8. Wilson, D. F. & Erecinska, M. (1972) Biomemhranes:
Molecular Arrangements and Transport Mechanisms
(van Deenan, L. L. M., Riemersma, J. C. & Tager, J.
M., eds) pp. 119- 132, North-Holland, Amsterdam.
9. Rosing, J. & Slater, E. C. (1972) Biochim. Biophys. Acta,
267,215- 290,
10. Wilson, D. F., Dutton, P. L. &Wagner, M. (1973) Curr.
Top. Bioenerget. 5,233-265.
11 Wilson, D. F. & Owen, C. (1973) Biochem. Biophys. Res.
Commun. 53,326- 333.
12 Wilson, D. F., Stubbs, M., Veech, R. L., Erecinska, M.
& Krebs, H. A. (1974) Biochem. J . 140, 57-64.
13 Rottenberg, H. (1970) Eur. J . Biochem. 15, 22-28.
14 Padan, E. & Rottenberg, H. (1973) Eur. J. Biochem. 40,
431 - 437.
15. Mitchell, P. & Moyle, J. (1967) Biochem. J . 104, 588

- 600.

Eur. J. Biochem. 50 (1974)

315

D. G. Nicholls
16. Azzone, G. F. & Massari, S. (1971) Eur. J . Biochem.
19,97 - 107.
17. Mitchell, P. & Moyle, J. (1969) Eur. J . Biochem. 9, 149
- 155.
18. Addanki, S., Cahill, F. D. & Sotos, J. F. (1967) Science
(Wash. D . C . ) 155, 1678- 1679.
19. Addanki, S., Cahill, F. D. & Sotos, J. F. (1968) J . Biol.
Chem. 243,2337-2348.
20. Hoek, J. B. Doctoral Thesis, University of Amsterdam
(1972).
21. Nicholls, D. G. & Lindberg, 0. (1972) FEBS Lett. 25,
61 64.
22. Rossi, E. & Azzone, G. F. (1968) Eur. J . Biochem. 7 ,
418-426.
23. Van Dam, K. & Tsou, C. S. (1969) in The Energy Level
~

and Metabolic Control in Mitochondria (Papa, S.,

Tager, J. M., Quagliarello, E. & Slater, E. C., eds)

pp. 21 - 29, Adriatica, Bari.
24. Chappell, J. B. & Crofts, A. R. (1966) in Regulation of
Metabolic Processes in Mitochondria (Tager, J. M.,
Papa, S., Quagliarello, E. & Slater, E. C., eds) pp. 229
- 314, Elsevier, Amsterdam.
25. Mitchell, P. & Moyle, J. (1967) Biochem. J . 105, 11471 162.

26. Chance, B. & Williams. G. R. (1956) Adv. Enzyme[. 17,

65-134.
27. Nordenbrand, K. & Ernster, L. (1973) Abstr. 9th In?.
Congr. Biochem. 236.
28. Liberman, E. A. & Skulachev, V. P. (1970) Biochim. Biophys. Acta, 216, 30-42.
29. Mitchell, P. & Moyle, J. (1968) Eur. J . Biochem. 4 , 530
- 539.

D. G. Nicholls, Department of Psychiatry, University of Dundee, Ninewells Hospital Medical School,

Dundee DD2 IUD. Great Britain

Eur. J. Biochem. 50 (1974)