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Effect of iodine on the electrical resistance of lipid bilayer membranes
Chem. Phys. Lipids 3 (1969) 98-101 © North-Holland Publ. Co., Amsterdam
EFFECT OF I O D I N E O N THE ELECTRICAL R E S I S T A N C E OF L I P I D BILAYER M E M B R A N E S
GORDON L. JENDRASIAK Radiation Laboratory *, University of Notre Dame, Notre Dame, Indiana 46556
Received 28 August 1967; resubmitted 17 September 1968 Iodine has been f o u n d to greatly lower the electrical resistance of lipid bilayer membranes 1, 2). This resistance lowering was found for membranes formed from egg lecithin ~,3) as well as for those formed from oxidized cholesterol2). Recently4), this lowering was found for membranes formed from egg lecithin-oxidized cholesterol ( 1 : l molar ratio), egg lecithincholesterol (1 : 1 molar ratio) and synthetic dipalmitoyl lecithin. The effect is independent o f the presence of ions such as N a + and Ca + + ; since dipalmitoyl lecithin membranes exhibit the lowering effect, the presence of a double bond in the lipid is unnecessary. In view of the nature of aqueous iodine solutions, the question arises as to whether 12 or 13 is the species responsible for the resistance lowering of lipid bilayer membranes. Experiments ~) indicate that for the egg lecithin membranes the resistance lowering may be due to I 2 rather than 13. In this work, however, the concentrations of iodine, [11], and triiodide, [I3], were varied simultaneously; moreover, the sum o f the concentrations of iodine, present as Iz and 13, was always equal to the concentration of a saturated solution of iodine in water. It thus seemed appropriate to study the effect of iodine, on the resistance of oxidized cholesterol membranes, with as great a variety of I2 and 13 concentrations as was experimentally feasible. Previous work s ) had indicated that the effect of iodine, in lowering the electrical resistance o f both oxidized cholesterol membranes and egg lecithin membranes, was quantitatively similar. Oxidized cholesterol membranes are, moreover, inherently more stable than egg lecithin membranes and, thus, lend themselves more readily to experimentation wherein they are exposed to a variety o f ionic environments. * The Radiation Laboratory of the University of Notre Dame is operated under contract with the U.S. Atomic Energy Commission. This is AEC document number COO-38-612.
ELECTRICAL RESISTANCE OF LIPID BILAYER MEMBRANES
99
Methods and material Solutions were prepared having a variety of concentrations of I2 and 13. The solutions were either 12 in H 2 0 , I 2 + N a I in H 2 0 or (CH3)aNI 3 in H20. HIO3 was used to lower the [I3]. The [I2] and [I3] values were determined spectroscopically using the extinction coefficient values for I2 and 13 as given in Awtrey6), 1951. Experiments were performed to assure that the resistance lowering was due only to I2 and/or I~ and not to the presence of other ions or to p H changes. Double distilled water was used for all solutions. Membranes were formed in these solutions from oxidized cholesterol dissolved in octane 7). The membranes were formed by the "brush technique" on a hole in a teflon cup. The resistance was determined by measuring the D.C. current through the membrane with a known voltage applied. The area of the membrane TABLE 1 Resistance of oxidized cholesterol membranes Row
[I21 ( M )
[Is ] ( M )
R(g2-
c m 2)
5.1 × 107
1 undetectable 2 3 4 5 6 7
1.1 7.4 1.1 9.7 8.0 1.0
x X × × x ×
10 z 10 -4 10 - s 10 4 10 4 10 a
8 9 10 11
4.8 x 10 -4 1.9xlO 4 2 . 0 x 10 -4 3.7×10 4
12 13 14 15 16 17
5.9 × 10 5 4.3 × 10 -5 4 . 7 × 10 5 9 . 0 × 10 -5 < 1 × 10 5 1.4×10 5
18 19 20 21
2.7 6.2 <2 <4
(< 3.8 1.2 8.3 1.5 1.0
10 -8) X 10 7 × 10 -5 x 10 5 × 10 -5 × 10 -4
× × × × x ×
104 104 103 103 103 102
10 -6 10 5 10 -5 10 4
4.8 × "8× -~ 6 × --~2×
10 a 10 2 10 2 10 z
x 10 8 × 10-7 × 10 -6 10 4 × 10 -4 × 10 - z
3.5 x 4.9 x 1.7 × <1 × ~ 8 × ~--2×
10 6 10 4 10 4 10 2 10 10
3.0 × 3.0× 4.9 × 1.0× 3.8 3.9 2.5 >7× 7.0 1.1
1.6 2.7 3.7 1.4 1.8 8.4
undetectable x × × x
10 6 10 -6 10 6 10 -6
(< 1.9 7.1 5.9
10 -8) × 10 -8 × 10 8 x 10 ~
3.3 6.4 7.9 2.3
× x × x
107 106 105 10 a
undetectable 22
( < 10 -7)
8.0 x 10 -6
7.8 × 10 4
100
GORDON L. JENDRASIAK
was determined with a microscope and associated micrometer disc. For each solution used, six to twelve membranes were formed; the resistance values were obtained five minutes after membrane formation. Table l summarizes the results of this work. The resistance values,/2, shown are average values. Results and discussion The results shown in table 1 indicate that when [I2] is approximately 1 × 10 - 3 M (row 2), a significant lowering of R is obtained even when no I 3 is detectable. The solubility of [2 in water (25°C) is about 1.3 x l 0 - 3 M. Increasing [13], when [[2] iS near its saturation value, lowers the resistance further, although not to as low a value as when less l 2 is present. Whether this possibly indicates an inhibition of the 13 by 12 is not clear at this time. As can be seen by examining row 17, lowering [I2] and making [I3] approximately 1 x l0 -a M results in the lowest/2 obtained. When [|2] is well below 1 x l 0 - 3 M , g seems to be dependent on [1~]; at a given [I2],/2 decreases as [I3] increases. Even when no I2 is detectable (row 22) but [I~-] is l0 - 6 - 10 -5 M, a large lowering of R is obtained. The value of/~ so obtained is about equal to that found with [I2] of 1 × 10 - 3 M and no 13 detectable. It has been proposed s) that a charge transfer interaction of lipid with l 2 leads to the resistance lowering observed in lipid bilayer membranes. The charge transfer interaction was proposed both for egg lecithin membranes as well as for oxidized cholesterol membranes. This charge transfer interaction for egg lecithin is thought to result in the formation of (lipid. I +) + I -. The I - then combines with free I2 to give 13. This scheme would account for the increase in I 3 concentration, as determined spectroscopically, upon addition of egg lecithin to an aqueous iodine solution. Because of spectroscopic similarities between iodine-egg lecithin solutions and iodine-oxidized cholesterol solutions, it has been speculated that a similar interaction might occur between iodine and oxidized cholesterol. If such a charge-transfer interaction does indeed occur with oxidized cholesterol and is responsible for the lowering of the membrane electrical resistance, one might expect the following: the addition of I 3, to a membrane whose resistance had already been lowered by iodine, should reverse the above reaction, provided a negligible amount of 12 w a s added simultaneously. The membrane resistance should thereby increase. When this experiment was performed with oxidized cholesterol membranes, however, the membrane resistance actually appeared to decrease slightly. The results shown in table 1 apparently differ from those of L~uger 3) and coworkers. It should be noted, however, that these workers used an A.C. measurement technique whereas the work reported here utilized a D.C.
ELECTRICAL RESISTANCE OF LIPID BILAYER MEMBRANES
101
method. L~iuger's membranes, moreover, were formed from egg lecithin whereas those used in obtaining the results of table 1 were formed from oxidized cholesterol. It would not be expected that this would account for the difference in results; indeed, egg lecithin membranes were studied in control experiments and were found to have the same D.C. resistance as did oxidized cholesterol membranes, when formed in solutions having the same [I2]/[I~] value. From the results of table l, it appears that I~ acts to lower the D.C. resistance of oxidized cholesterol membranes. 12 may also act to lower the membrane resistance although not necessarily by the same mechanism as does I~. The I~, moreover, seems to produce a given resistance decrease at much lower concentrations than does I2. Whether the mechanism of resistance lowering, by iodine, is the same for both oxidized cholesterol and egg lecithin membranes is not certain at this time. The fact that both types of membranes exhibit similar R values, when they are formed in solutions having the same [I2]/[I~] ratio, suggests that the mechanism may well be the same. The fact that I ; is quite effective in lowering the electrical resistance of oxidized cholesterol membranes does not in itself help to clarify the mechanism of charge conduction in the membranes. Electronic conduction, as has been suggested by L~iugerl), might indeed occur with I~- acting as an electron donor at the membrane-water interface. On the other hand, I~may well interact with the lipid membrane thereby altering its structure and rendering the membrane more permeable to ions or protons. The paucity of experimental data concerning both membrane structure and the nature of the charge carrier, through the membrane, does not allow a choice to be made, at present, between these two possibilities. References
1) P. L~iuger, W. Lesslauer, E. Marti and J. Richter, Biochim. Biophys. Acta, 135 (1967) 20 2) B. Rosenberg and G. L. Jendrasiak, Chem. Phys. Lipids 2 (1968) 47 3) P. L/iuger, J. Richter and W. Lesslauer, Ber. Bunsenges. Phys. Chem. 71 (1967) 906 4) G. L. Jendrasiak, unpublished results (1968) 5) G. L. Jendrasiak, Ph.D. Thesis, Michigan State University (1967); also published as report AD 657954 for Defense Documentation Center, Alexandria, Va. 6) A. D. Awtrey and R. E. Connick, J. Am. Chem. Soc. 73 (1951) 1842 7) H. Tien and S. Carbone, Nature 212 (1966) 718 8) B. Bhowmik, G. L. Jendrasiak and B. Rosenberg, Nature 215 (1967) 842
EFFECT OF I O D I N E O N THE ELECTRICAL R E S I S T A N C E OF L I P I D BILAYER M E M B R A N E S
GORDON L. JENDRASIAK Radiation Laboratory *, University of Notre Dame, Notre Dame, Indiana 46556
Received 28 August 1967; resubmitted 17 September 1968 Iodine has been f o u n d to greatly lower the electrical resistance of lipid bilayer membranes 1, 2). This resistance lowering was found for membranes formed from egg lecithin ~,3) as well as for those formed from oxidized cholesterol2). Recently4), this lowering was found for membranes formed from egg lecithin-oxidized cholesterol ( 1 : l molar ratio), egg lecithincholesterol (1 : 1 molar ratio) and synthetic dipalmitoyl lecithin. The effect is independent o f the presence of ions such as N a + and Ca + + ; since dipalmitoyl lecithin membranes exhibit the lowering effect, the presence of a double bond in the lipid is unnecessary. In view of the nature of aqueous iodine solutions, the question arises as to whether 12 or 13 is the species responsible for the resistance lowering of lipid bilayer membranes. Experiments ~) indicate that for the egg lecithin membranes the resistance lowering may be due to I 2 rather than 13. In this work, however, the concentrations of iodine, [11], and triiodide, [I3], were varied simultaneously; moreover, the sum o f the concentrations of iodine, present as Iz and 13, was always equal to the concentration of a saturated solution of iodine in water. It thus seemed appropriate to study the effect of iodine, on the resistance of oxidized cholesterol membranes, with as great a variety of I2 and 13 concentrations as was experimentally feasible. Previous work s ) had indicated that the effect of iodine, in lowering the electrical resistance o f both oxidized cholesterol membranes and egg lecithin membranes, was quantitatively similar. Oxidized cholesterol membranes are, moreover, inherently more stable than egg lecithin membranes and, thus, lend themselves more readily to experimentation wherein they are exposed to a variety o f ionic environments. * The Radiation Laboratory of the University of Notre Dame is operated under contract with the U.S. Atomic Energy Commission. This is AEC document number COO-38-612.
ELECTRICAL RESISTANCE OF LIPID BILAYER MEMBRANES
99
Methods and material Solutions were prepared having a variety of concentrations of I2 and 13. The solutions were either 12 in H 2 0 , I 2 + N a I in H 2 0 or (CH3)aNI 3 in H20. HIO3 was used to lower the [I3]. The [I2] and [I3] values were determined spectroscopically using the extinction coefficient values for I2 and 13 as given in Awtrey6), 1951. Experiments were performed to assure that the resistance lowering was due only to I2 and/or I~ and not to the presence of other ions or to p H changes. Double distilled water was used for all solutions. Membranes were formed in these solutions from oxidized cholesterol dissolved in octane 7). The membranes were formed by the "brush technique" on a hole in a teflon cup. The resistance was determined by measuring the D.C. current through the membrane with a known voltage applied. The area of the membrane TABLE 1 Resistance of oxidized cholesterol membranes Row
[I21 ( M )
[Is ] ( M )
R(g2-
c m 2)
5.1 × 107
1 undetectable 2 3 4 5 6 7
1.1 7.4 1.1 9.7 8.0 1.0
x X × × x ×
10 z 10 -4 10 - s 10 4 10 4 10 a
8 9 10 11
4.8 x 10 -4 1.9xlO 4 2 . 0 x 10 -4 3.7×10 4
12 13 14 15 16 17
5.9 × 10 5 4.3 × 10 -5 4 . 7 × 10 5 9 . 0 × 10 -5 < 1 × 10 5 1.4×10 5
18 19 20 21
2.7 6.2 <2 <4
(< 3.8 1.2 8.3 1.5 1.0
10 -8) X 10 7 × 10 -5 x 10 5 × 10 -5 × 10 -4
× × × × x ×
104 104 103 103 103 102
10 -6 10 5 10 -5 10 4
4.8 × "8× -~ 6 × --~2×
10 a 10 2 10 2 10 z
x 10 8 × 10-7 × 10 -6 10 4 × 10 -4 × 10 - z
3.5 x 4.9 x 1.7 × <1 × ~ 8 × ~--2×
10 6 10 4 10 4 10 2 10 10
3.0 × 3.0× 4.9 × 1.0× 3.8 3.9 2.5 >7× 7.0 1.1
1.6 2.7 3.7 1.4 1.8 8.4
undetectable x × × x
10 6 10 -6 10 6 10 -6
(< 1.9 7.1 5.9
10 -8) × 10 -8 × 10 8 x 10 ~
3.3 6.4 7.9 2.3
× x × x
107 106 105 10 a
undetectable 22
( < 10 -7)
8.0 x 10 -6
7.8 × 10 4
100
GORDON L. JENDRASIAK
was determined with a microscope and associated micrometer disc. For each solution used, six to twelve membranes were formed; the resistance values were obtained five minutes after membrane formation. Table l summarizes the results of this work. The resistance values,/2, shown are average values. Results and discussion The results shown in table 1 indicate that when [I2] is approximately 1 × 10 - 3 M (row 2), a significant lowering of R is obtained even when no I 3 is detectable. The solubility of [2 in water (25°C) is about 1.3 x l 0 - 3 M. Increasing [13], when [[2] iS near its saturation value, lowers the resistance further, although not to as low a value as when less l 2 is present. Whether this possibly indicates an inhibition of the 13 by 12 is not clear at this time. As can be seen by examining row 17, lowering [I2] and making [I3] approximately 1 x l0 -a M results in the lowest/2 obtained. When [|2] is well below 1 x l 0 - 3 M , g seems to be dependent on [1~]; at a given [I2],/2 decreases as [I3] increases. Even when no I2 is detectable (row 22) but [I~-] is l0 - 6 - 10 -5 M, a large lowering of R is obtained. The value of/~ so obtained is about equal to that found with [I2] of 1 × 10 - 3 M and no 13 detectable. It has been proposed s) that a charge transfer interaction of lipid with l 2 leads to the resistance lowering observed in lipid bilayer membranes. The charge transfer interaction was proposed both for egg lecithin membranes as well as for oxidized cholesterol membranes. This charge transfer interaction for egg lecithin is thought to result in the formation of (lipid. I +) + I -. The I - then combines with free I2 to give 13. This scheme would account for the increase in I 3 concentration, as determined spectroscopically, upon addition of egg lecithin to an aqueous iodine solution. Because of spectroscopic similarities between iodine-egg lecithin solutions and iodine-oxidized cholesterol solutions, it has been speculated that a similar interaction might occur between iodine and oxidized cholesterol. If such a charge-transfer interaction does indeed occur with oxidized cholesterol and is responsible for the lowering of the membrane electrical resistance, one might expect the following: the addition of I 3, to a membrane whose resistance had already been lowered by iodine, should reverse the above reaction, provided a negligible amount of 12 w a s added simultaneously. The membrane resistance should thereby increase. When this experiment was performed with oxidized cholesterol membranes, however, the membrane resistance actually appeared to decrease slightly. The results shown in table 1 apparently differ from those of L~uger 3) and coworkers. It should be noted, however, that these workers used an A.C. measurement technique whereas the work reported here utilized a D.C.
ELECTRICAL RESISTANCE OF LIPID BILAYER MEMBRANES
101
method. L~iuger's membranes, moreover, were formed from egg lecithin whereas those used in obtaining the results of table 1 were formed from oxidized cholesterol. It would not be expected that this would account for the difference in results; indeed, egg lecithin membranes were studied in control experiments and were found to have the same D.C. resistance as did oxidized cholesterol membranes, when formed in solutions having the same [I2]/[I~] value. From the results of table l, it appears that I~ acts to lower the D.C. resistance of oxidized cholesterol membranes. 12 may also act to lower the membrane resistance although not necessarily by the same mechanism as does I~. The I~, moreover, seems to produce a given resistance decrease at much lower concentrations than does I2. Whether the mechanism of resistance lowering, by iodine, is the same for both oxidized cholesterol and egg lecithin membranes is not certain at this time. The fact that both types of membranes exhibit similar R values, when they are formed in solutions having the same [I2]/[I~] ratio, suggests that the mechanism may well be the same. The fact that I ; is quite effective in lowering the electrical resistance of oxidized cholesterol membranes does not in itself help to clarify the mechanism of charge conduction in the membranes. Electronic conduction, as has been suggested by L~iugerl), might indeed occur with I~- acting as an electron donor at the membrane-water interface. On the other hand, I~may well interact with the lipid membrane thereby altering its structure and rendering the membrane more permeable to ions or protons. The paucity of experimental data concerning both membrane structure and the nature of the charge carrier, through the membrane, does not allow a choice to be made, at present, between these two possibilities. References
1) P. L~iuger, W. Lesslauer, E. Marti and J. Richter, Biochim. Biophys. Acta, 135 (1967) 20 2) B. Rosenberg and G. L. Jendrasiak, Chem. Phys. Lipids 2 (1968) 47 3) P. L/iuger, J. Richter and W. Lesslauer, Ber. Bunsenges. Phys. Chem. 71 (1967) 906 4) G. L. Jendrasiak, unpublished results (1968) 5) G. L. Jendrasiak, Ph.D. Thesis, Michigan State University (1967); also published as report AD 657954 for Defense Documentation Center, Alexandria, Va. 6) A. D. Awtrey and R. E. Connick, J. Am. Chem. Soc. 73 (1951) 1842 7) H. Tien and S. Carbone, Nature 212 (1966) 718 8) B. Bhowmik, G. L. Jendrasiak and B. Rosenberg, Nature 215 (1967) 842