I24
POTENTIAL DIFFERENCES ACROSS NATURAL
MEMBRANES SEPARATING UNLIKE SALT
SOLUTIONS
BY S. C. BROOKS, A. C. GIESE AND R. I. GIESE.
(From Dept. of Zoology, Univ. of California, and Hopkins Marine Station,
Stanford University.)
(Received nth September, 1930.)
(With Two Text-figures.)
(1911) showed that when the cutinised epidermis of the apple
separates two unlike solutions of electrolytes, a potential difference develops, which
they interpret as being due to exchange of ions, mainly cations, across the epidermis. They suppose that the faster-moving ions tend to penetrate more rapidly
through the epidermis, and hence build up a potential difference.
Michaelis (1925) gives a mathematical development of essentially this theory to
explain his own experiments with "dried" celloidin membranes which are permeable to cations but not to anions, and which, like the apple epidermis, develop
a potential difference which is constant for a considerable period of time.
Recently, Mond and Hoffmann (1928) described a membrane made of celloidin
and Rhodamin B, which is selectively anion permeable. This was followed by
Hober and Hoffmann's (1928) study of a mosaic membrane made up of alternating
areas of cation permeable celloidin and anion permeable celloidin-Rhodamin B. In
this case the potential difference obtained was considerably lower than that for
either the celloidin or celloidin-Rhodamin B membranes separately, the potential
differences of the two areas being in opposite directions and tending to annul one
another.
The cutinised epidermis from the inner surface of bulb scales of the onion resembles these membranes in that there is no diffusion of ions through the epidermis
from solutions of electrolytes into distilled water (Brooks, 1917).
It, therefore, seemed desirable to study the nature of the potential differences
developed by the onion epidermis when it separates two solutions containing ions,
with a view to further analysis of the electromotive mechanisms at work.
LOEB AND BEUTNER
Potential Differences across Natural Membranes
125
METHOD.
The membrane was stretched as a partition between two half cells connected to
saturated calomel electrodes by saturated KC1 capillary stop-cock bridges (Fig. 1).
The potential differences were determined by the use of a potentiometer with an
accuracy of about 0-5 mv. To avoid leakage, the ground edges of the cells were
greased with purified wax vaseline, and the membrane was then carefully and tightly
fitted around. The cells were insulated by a glass plate and both cells and plate were
kept dry throughout the experiments. The bridges and the calomel electrodes were
checked to ensure that no potential difference was being developed except at the
membrane, or at the liquid junctions between the capillary stop-cock bridges and
the solutions in the half cells. The latter may be assumed to be negligible in comparison to the potential difference developed at the membrane except, possibly, in
C
— C
Fig. 1. Half cells and membrane as used for the measurement of potential differences. M: membrane separating solutions S1 and <S2; R, rubber band holding half cells together; C, C, capillary bridges
to calomel electrodes.
the case of the concentration potential difference with CaCl2, and of the chemical
potential differences with common cation and different anion. Even in these cases
it is not possible to explain the observed phenomena as liquid junction potentials.
The bridges were refilled before each experiment, and the ends washed in the
solutions to be used. When solutions were changed, the cells and membrane were
rinsed with the solutions to be used.
RESULTS.
Table I gives the data for the experiments on concentration chains. The potential difference of concentration chains composed of o-iMKCL/membrane/o-oiM
KC1 rose shortly after the initial reading, was fairly constant for 15 to 45 minutes,
then gradually fell off during the course of 24 hours or more. The behaviour of
similar concentration chains using NaCl or LiCl was similar (see Fig. 2).
It is this plateau region of constant potential difference that we consider the
quasi-equilibrium, and refer to in our experiments. It is reproducible within a few
JEB-Vlllii
9
126
S. C. BROOKS, A. C. G I E S E and R. I. GIESE
millivolts for each membrane when the cells are refilled with fresh solution, but
quite individual to each membrane. The fall in potential difference was not due to
tearing, since membranes artificially punctured with very fine glass needles showed
a sudden steep drop in potential difference. This test also makes it clear that the
membrane itself is the principal source of the potential difference.
Table I. Concentration potentials obtained with O - I M / O - O I M solutions of KCl,
NaCl, LiCl, and CaCl2. The sign of the potential is that of the o-oiM solution, the
positive current tending to flow from the positive solution through the potentiometer.
KCl
Membrane
Potential
difference
Membrane
No.
mv.
12
44-2
34-5
18-0
14
15
23
26
27
36
48
55
63
65
67
68
404
40-1
32-5
32-5
391
198
37-i
29-6
17-1
139
71
120
74
78
216
80
81
92
230
93
94
LiCl
NaCl
16-1
15-3
38-5
386
42-4
+29-2
Mean
Probable error
±i-5
Potential
difference
Membrane
No.
mv.
11
12
280
366
335
33'3
34'5
335
18
19
16
23
26
3°
32
289
36
48
67
22-5
320
9'4
2I'O
20-7
2O-7
I2-I
24-1
15-0
33-2
70
72
74
£
81
93
94
392
CaCl2
Potential
difference
Membrane
Potential
difference
No.
mv.
No.
mv.
12
39-8
10-5
213
306
21
23
34
44
45
48
67
u
-4'3
-3-6
±0
— I-I
-6-2
±0
—2-2
-1-5
±0
±O
-7-5
-135
±0
— 129
68
70
72
72
357
23-2
22-4
15-5
22-6
4
T,
80
8-6
73
81
70
72
68
12-3
20-3
22-5
93
93
94
74
2I-I
80
81
93
94
22-9
15-7
321
94
38-9
8
+25-7
+ 23-1
-3
±1-4
±1 4
±2 4
The maximum value for KCl was 44-2 mv., the majority of the readings fell
between 20 and 40 mv. and a few were as low as 12 mv. The mean value for KCl
concentration chains was 29-2 mv., for NaCl 25*7 mv., and for LiCl 23-1 mv.
The difference between the mean potential differences developed by KCl and
LiCl is nearly three times the probable error of the difference. This may be taken as
a significant difference, the intermediate position of NaCl in itself not differing
significantly from KCl on the one hand or LiCl on the other, is thus to be expected
on the basis of the physical characteristics of the ions in question. It seems probable
that the potential differences developed by concentration chains of these salts are
really of the relative magnitudes given. In order of descending potential difference
we would then have K > Na > Li.
Similar concentration chains using CaCl2 showed somewhat similar behaviour
when a measurable potential was developed at all, but the potential differences were
Potential Differences across Natural Membranes
127
at best very small and were opposite in sign to those observed with alkali metal
chlorides. The mean potential difference does not greatly exceed its own probable
error but its consistently negative sign is suggestive.
Table II gives the concentration potential differences observed when different
salts were studied one after another with one and the same membrane in the case of
eight different membranes. The readings were made in the sequence indicated,
except that the CaCl2 solution was studied after the others because of an effect on
the membrane which will be discussed later.
+42
+ 35
•0
~NaCT
•§ +28
w~--—.
N
/
.3 +21
i
i d (48)
I
\
+ 7
\
KC1(14)
+14
\
\
s.
\\
v
\
\
\\
Of,
u
<§~— --«Ca
:i 2 (80)
CaCl2 (74)
20
40
60
Time in minutes
80
100
120
1 1
20
29
38
Time in hours
Fig. 2. Changes with time of the observed potential differences between o-iM and o-oiM solutions
of the indicated salts separated by membranes of onion epidermis. The numbers of the individual
membranes correspond to those given in Table I.
When like concentrations of different salts are used on the two sides of the membrane, a potential difference may arise which Michaelis has called a "chemical"
potential difference in contradistinction to the "concentration" potential difference
discussed above. One type of chemical potential difference was observed when
equally concentrated solutions of chlorides with different cations were separated by
onion epidermis. The results of these series agree in general with the lyotropic
series, the potential differences for the different ions decreasing in the order
K > Na > Li. This same order was found by Michaelis in the case of dried celloidin membranes. It will be noted that the potential difference with KC1 against
LiCl of the same concentration is the largest and, within the limits of error, equal
to the sum of the potential difference for KC1: NaCl and that for NaCl: LiCl.
9-2
128
S. C. BROOKS, A. C. G I E S E and R. I. G I E S E
The other type of chemical potential difference was observed when o-iM KCl
was used on the one side of the membrane and o-iM KBr, KI, KNO 3 , K J S O J or
KSCN on the other. These potential differences were so low as to be almost
negligible. While, in the case of cation potential differences, all the mean values
are significantly greater than their probable errors, in the case of the anion potential
differences this is not the case. Only one membrane (No. 63) consistently showed a
potential difference in the case of all the salts. Several membranes showed potential
differences in the case of Cl': N0 3 ' and Cl': SO4" (see Table II).
Table I I . Concentration, cation and anion potential differences exhibited by individual
membranes. Unless otherwise stated the figures indicate the potential difference in mv.,
the solution given last being positive to the first.
Set up
Potential differences
Concentration
potential
differences
01 :ooiM
Numbers of membranes
48
63
67
74
80
8l
93
40-4
33-3
35-7
39-i
320
22-6
371
171
94
8-6
-4'3
21-6
20-7
2I-I
-2-2
23-0
24-1
229
IS-3
150
iS-7
±OO
38-4
33-2
321
-7-5
29-0
24-0
22-7
-31
±2-6
±30
±2-4
±0-9
60
59
2-7
3-7
2-3
4-S
2-S
i-6
i-7
8-S
4-3
47
60
7-8
4-3
±o-8
2-O
I'l
±o- S
4-2
23
10
36
o-6
±00
i-8
±00
±00
±00
±00
i-5
1-5
±o-o
o-6
16
o-S
3-6
7-1
70
±00
33
242
274
3S'2
12-2
2-O
2-S
2I-2
149
O O O O O
±o-o
±00
±00
±00
±00
o-S
H-H-H-H-H-
±00
O O O O O
: KBr
: KI
: KNO,
: KSCN
: iK8SO4
66 66 6
KCl
KCl
KCl
KCl
KCl
H-H-H-H-H-
Anion potential
difference
o-i : o-iM
0-3
±00
09
i-o
i-4
363
24-1
H-
14-1
104
2-8
0 p 0 0 0
95
5-0
4-S
60
99
5-5
3-2
-i-s
H-H-
Cation potential
difference
o-i : O'lM
KCl : LiCl
KCl : NaCl
NaCl : LiCl
KCl : CaCl2
23
6 6 6 6 6
KCl : KCl
NaCl : NaCl
LiCl : LiCl
CaCl2 : CaCla
Mean Probable
error
±0-7
±0-3
±0-7
(±o-s)
±0-3
±o-6
±o-6
Check concentration potential
difference
o-i : O'OiM
KCl : KCl
21-2
±I-I
Apparently the cation potential difference is established at once and, except for
irregular minor fluctuations, there is little subsequent change. As the anions developed almost no potential differences, the rate at which equilibrium is established
in such systems cannot be discerned from the data at hand.
The solutions used have some effects on the membranes, as shown by the relative values of the concentration potential difference for KCl at the beginning and
at the end of a series (see bottom line, Table II). This was particularly noticeable in
Potential Differences across Natural Membranes
12()
the case of CaCl2, which so affected the membrane that the potential difference for
the KCl concentration chain, observed immediately after CaCl2 had been in contact
with the epidermis, was considerably lower than before, and returned to the initial
value only after several washings. CaCl2 was applied first on the cuticular side, then
on the cellular side of the epidermis, and the effect was found to be more pronounced in the latter case. Likewise o-iM CaCl2 on the cellular side was found to
lower the potential difference more than o-oiMCaCl2. Washing with o-iM KCl
gradually dissipated the effect, which is, therefore, reversible (see Tables III and IV).
Table I I I . Effects of calcium on the concentration potential difference for KCl.
o-iMCaCl 2
applied to
surface indicated
Solution applied
to other surface
Cuticular
Cuticular
Cuticular
Cuticular
Cellular
Cellular
Cellular
Cellular
Cellular
KClo-iM
KClo-iM
CaCl2 o-oiM
CaCl 2 ooiM
KCI01M
KClo-iM
KCI01M
CaCls o-oiM
CaCl2 001M
No. of
membrane
85
89
81
93
85
88
90
93
94
Equilibrium concentration
potential difference
for KCl
Before Ca
After Ca
2O-8
2S-8
21-7
26-0
15-2
22-7
3O-O
299
13-7
314
61
24-4
210
3IO
326
28-s
34°
210
Table IV. Effects of renewing solutions of KCl on the concentration potential difference
for KCl after the cellular side of the epidermis has been exposed to o-iM CaCl^for
3 to 53 minutes.
Concentration potential difference for KCl after
application of CaCl2
No. of
membrane
82
85
88
9°
91
93
94
With first KCl solution
(After renewing solution number of
times indicated.) (Equilibrium values)
Initial
Equilibrium
1
2
3
0
20
61
244
2I-O
2 3 -2
2I-O
IO-2
I5-O
162
17-8
2-6
6-S
3-8
19-7
28-s
28-s
338
29-0
318
26-7
336
130
S. C. BROOKS, A. C. G I E S E and R. I. G I E S E
DISCUSSION.
The rise and temporary quasi-equilibrium in concentration potential differences
may be interpreted as indicating that the lower epidermis of the onion scale is permeable to the univalent cations tested, but perhaps not to Ca-. The gradual falling
off in potential difference might be due to the slow establishment of an anion quasiequilibrium which opposed its potential difference to that of the cation quasiequilibrium. If the membranes were permeable to anions as well as to cations, it
was to be expected that the final resultant quasi-equilibrium potential difference
would be lower than 57 mv., the value calculated on the basis of complete impermeability to ions of one sign of charge. Furthermore, if the quasi-equilibrium for
ions of one sign were established sooner than that for ions of opposite sign, the
potential difference would pass through a maximum and then fall off with increasing
time. The resemblance of this behaviour to that of mosaic membranes has been
mentioned above.
Different specimens of onion epidermis exhibit a wide variety of behaviour.
Some appear to be very impermeable to anions, so that the cations build up a high
potential difference which is maintained for 1 or more hours; in most cases anions
are probably penetrating the membrane and setting up their quasi-equilibria relatively soon, so that a relatively low and brief potential difference results; a few
membranes appear to have so high a permeability to anions that the potential difference is abnormally low and brief (Table I).
If the membrane were permeable to anions, anion chemical potentials might be
expected. Some membranes show such potential differences, but they seem usually
to be small or lacking (see Table II). However, this may not be due entirely to lack
of permeability to anions. The relative mobilities of the anions used differ much less
among themselves than do those of the cations used. It seems quite possible that
the hindrance offered to similar ions by membranes of this type is related to their
mobilities in dilute aqueous solution. In this event the anion potential differences
might well be much smaller than those due to the cations.
The concentration potential difference given by CaCl2 yields a further insight
into the situation. The reversal of sign of the potential difference, as compared with
that for a similar KC1 chain, can most easily be explained by assuming that the
epidermis is more permeable to Cl' than to Ca-. Under the conditions of the
experiments the diffusion potential due to Ca- would be opposite in sign to the
potential difference actually observed. The diffusion potential due to Cl' would be
of the same sign as that observed, and in view of the sign of the observed potential
difference, it must equal or exceed that due to calcium.
If Cl' penetrates the epidermis, the other anions used must do so also, otherwise
chemical potential differences would be built up when different salts of the same
cation are present in equal concentrations on opposite sides of the membrane. Since
little or no potential difference was actually found, the epidermis must be about
equally permeable to the different anions.
Potential Differences across Natural Membranes
131
That the living protoplasm plays some part in producing these potential differences is suggested by those experiments (Table III) in which the same membranes
were used for a series of observations with different salts including CaCl2. In these
experiments CaCl2 was found to cause a reversible decrease in the observed concentration potential difference of KC1. Since the effect was more marked when the
CaCl2 was on the cellular side, it must be due to the action of Ca-- on either protoplasm or cell-wall material. Either of these would be more or less protected from
CaCl2 applied to the cuticular side of the epidermis, since it is probably the cuticle
itself which is least permeable to ions (Brooks, 1917).
That the potential differences are not primarily due to living protoplasm is
shown by the fact that dead membranes behaved like living ones, though the observed potential differences were lower. However, a satisfactory method of killing
the cells of the membrane was not devised, and further discussion of the part played
by the living protoplasm must be left to the future.
The effect of Ca-- might be attributed to the formation of calcium pectinates,
with a resultant change in the physical state of part of the cell-wall material; or it
might be attributed to a decrease in the permeability of the protoplasm which quite
probably occurs. Whatever the nature of the effect, it is reversible in nature, as
is shown by the progressive increase in the concentration potential difference
of KC1 following successive washings of the epidermis with a KC1 solution
(Table IV).
The lower epidermis of the scales of the onion seems, then, to be permeable to
cations, and, at least to a limited degree, to anions. Since salts cannot pass through
these membranes into distilled water (Brooks, 1917), the facts are most easily interpreted by supposing the onion epidermis to embody a sort of mosaic of selectively
ion permeable areas of the nature of the patch membrane described by Hober and
Hoffmann (1928).
These experiments suggest that bioelectric potentials observed in other plant
cells may also be due to the properties of the cell walls as well as of the protoplasm.
It can hardly be considered safe to assume without special study that such potentials
are due to the protoplasm alone.
SUMMARY.
1. When the lower epidermis of the bulb scale of the onion separates o-iM and
o-oiM solutions of KC1, NaCl, or LiCl a transient "concentration" potential
difference was observed, whose magnitude decreased in the order: K > Na > Li.
2. The potential difference of o-xM: o-oiM CaCl2 is small and opposite in sign
to that of the above chains.
3. When the epidermis separates like concentrations of chlorides of K, Na, and
Li a cation "chemical" potential difference arises which is low but steady.
4. When K-salts with different anions are present on the opposite sides of the
membrane, the anion "chemical" potential difference is small or lacking.
132
S. C. BROOKS, A. C. G I E S E and R. I.
GIESE
5. These facts are most simply accounted for by assuming that the epidermis is
a mosaic of cation and anion permeable areas, the permeability to alkali metal
cations much exceeding that for calcium ion or the anions tried.
6. It is pointed out that the cell walls may perhaps participate in the production
of the bioelectric potentials observed in other plants.
REFERENCES.
BROOKS, S. C. (1917). Bot. Gaz. 64, 509.
H6BER, R. and HOFFMANN, F . (1928). Arch. Ges. Physiol. 220, 558.
LOEB, J. and BEUTNER, R. (1911). Science, 34, 884.
MICHAELIS, L. (1925). Joum. Gen. Physiol. 8, 23.
MOND, R. and HOFFMANN, F . (1928). Arch. Ges. Physiol. 220, 194.