[40]
D E S I G N , PROPERTIES, A N D A P P L I C A T I O N S
439
[40] D e s i g n , P r o p e r t i e s , a n d A p p l i c a t i o n s o f N e u t r a l Ionophores By WILHELM SIMON and ERNESTO CARAFOLI Synthetic Neutral Ionophores Design Features. Electrically neutral, lipophilic ion-complexing agents of rather small relative molar mass are known to behave as ionophores or ion carriers. 1 They have the capability to selectively extract ions from aqueous solutions into a hydrophobic membrane phase and to transport these ions across such barriers by carrier translocation. In order for a ligand to behave as an ionophore for cations, several aspects have to be considered (see also Simon et al.2). a. A carrier molecule should be composed of polar and nonpolar groups. b. The carder should be able to assume a stable conformation that provides a cavity, surrounded by the polar groups, suitable for the uptake of a cation, while the nonpolar groups form a lipophilic shell around the coordination sphere. These groups must ensure sufficiently large lipid solubility for ligand and complex. This is one reason why classic electrically charged complexing agents, such as ethylenediamine tetraacetic acid (EDTA), will not behave as carders in membrane systems. c. Among the polar groups of the ligand sphere, there should be preferably five to eight, but not more than twelve coordinating sites, such as oxygen atoms. d. High selectivities are achieved by locking the coordinating sites into a rigid arrangement around the cavity. Such rigidity can be enhanced by the presence of bridged structures or hydrogen bonds. Within one group of the periodic system, the cation that best fits into the offered cavity is preferred. Ideally, all cations should be forced into accepting the same given number of coordinating groups. e. Notwithstanding requirement (d), the ligand should be flexible enough to allow a sufficiently fast ion exchange. This is possible only with a stepwise substitution of the solvent molecules by the ligand groups. Thus, a compromise between stability (d) and exchange rate (e) has to be found. 1Yu. A. Ovchinnikov, V. T. Ivanov, and A. M. Shkrob, BBA Libr. 12, 1 (1974). 2 W. Simon, W. E. Morf, and D. Ammann, in "'Calcium Binding Proteins and Calcium Function" (R. H. Wasserman et al., eds.), p. 50. North-Holland Publ., Amsterdam, 1977.
METHODS IN ENZYMOLOGY,VOL. LVI
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6
440
SPECIALIZED TECHNIQUES
[40]
f. To guarantee an adequate mobility, the overall dimensions of a carrier should be rather small but still compatible with high lipid solubility. Attractive binding sites are ligand atoms which are capable of competing with water molecules in the complexation of ions. Through variation of molecular parameters, such as dipole moment and polarizability of the binding sites as well as the van der Waals radius of the ligand atoms, the interaction selectivity may be considerably influenced (for details, see Simon et al.2"3). It is a well known fact that complexes of polydentate ligands have enhanced stability over their unidentate counterparts (chelate effect), and complexes of macrocyclic ligands are, as a rule, more stable than those of noncyclic polydentate ligands (macrocyclic effect). 3,4 According to Adamson,5 the chelate effect is largely a consequence of the asymmetry of the standard reference state. Stabilization effects beyond what has been discussed may be due to a reduction in translational and/or rotational entropy of the free polydentate ligand. In macrocyclic and especially in macropolycyclic ligands, repulsions, e.g. between binding sites, are already built in and, in addition, optimal solvation of the free ligand sites may be prevented so that the formation of the complexes is further favored.4 Due to favoured conformations of a multidentate ligand of a given constitution, the coordination shell (i.e., coordination number and cavity radius) may be predetermined and therefore selectivity can be induced even by non-macrocyclic molecules. Indeed, the non-macrocyclic neutral carriers shown in Fig. 1 are capable of inducing extremely high selectivity for cations in certain membranes. 6 The ligands ETH 1001r (Fluka AG, Buchs, Switzerland) and ETH 1293 exhibit rather high selectivity for Ca z÷. ETH 1001 and ETH 129 may form 1:2 and 1:3 Ca2+-ligand complexes, respectively. Since the ether and amide oxygen atoms participate in the coordination of the calcium ion, there will be a coordination sphere of 8 oxygen atoms for ETH 1001 and of 9 oxygen atoms for ETH 129. In both cases the oxygen-Ca 2÷ distance is a W. E. Moff, R. Bissig, D. Ammann, E. Pretsch, and W. Simmon, in "Multidentate Macrocyclic Molecules" (J. J. Christensen and R. M. Izatt, eds.). Wiley (Interscience), New York (in press). 4 J.-M. Lehn, Struct. Bonding (Berlin), 16, 1 (1973). 5 A. W. Adamson, J. A m . Chem. Soc. 76, 1578 (1954). D. Ammann, R. Bissig, Z. Cimerman, U. Fiedler, M. Giiggi, W. E. Morf, M. Oehme, H. Osswald, E. Pretsch, and W. Simon, in "Ion and Enzyme Electrodes in Biology and Medicine" (M. Kessler, et al., eds.), p. 22. Urban & Schwarzenberg, Munich, 1976. 7 D. Ammann, M. Giiggi, E. Pretsch, and W. Simon, Anal. Lett. 8, 709 (1975).
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
441
around 2.4 A, which roughly corresponds to the sum of the radii of oxygen and Ca2+.3'8'9 The synthetic ionophores ETH 1097,1° ETH 227,11 and ETH 1576 are capable of inducing selectivity for Na ÷ over K ÷ whereas ETH 149TM has interesting selectivity for Li+. 6,1z Applications of Neutral Ionophores
Ion Selective Electrodes, General.13 The ionophores shown in Fig. 1 may be used as components in liquid membrand electrodes for the measurement of ion activities. 6 Ideally, such an electrode cell assembly (Fig. 2) generates an electric potential difference or emf that is proportional to the logarithm of the activity of the ion to be measured (Nernstian response). In the classic arrangement, the ion selective membrane consisted of a filter paper impregnated with a water-immiscible solution of the ion exchanger. Significant, especially mechanical, improvements were obtained by incorporating the electroactive material into polyvinylchloride (PVC). For the preparation of microelectrodes with tip diameters of around 1/zm, the ion selective liquid may be placed directly into a capillary between the sample solution and the internal filling solution (see Fig. 2). Because of the extremely high selectivity of the Ca2+-ionophore ETH 1001,13 and the interest in the measurement of Ca 2+ in biological systems, we will focus here on the determination of the activity aca using ion selective electrodes (for further details, see Simon et al. 13). A semiempirical and fairly successful approach, which has been recommended by the IUPAC, to treat real membrane electrode systems is offered by the extended Nicolsky-Eisenman equation: E
g~a +s
log (aca "~ ~ g~aatM (aM) 2tz" /)
(1)
M
where E is the cell potential (emf), E~a the reference potential at constant temperature; s the slope of electrode response function, aca the activity of primary ion Ca 2+ in sample solution, a~ the activity of an interfering ion M (charge zM) in sample solution, g cPot a M the potentiometric selectivity coefficient in respect to ion M. If there is no interference by ions other than 8 K. Neupert-Laves and M. Dobler, Helv. Chim. Acta 60, 1861 (1977) M. Dobler, private communication. 10 D. Ammann, E. Pretsch, and W. Simon, Anal. Lett. 7, 23 (1974). 11M. Giiggi, M. Oehme, E. Pretsch, and W. Simon, Helv. Chim. Acta 59, 2417 (1976). 12N. N. L, Kirsch, R. J. Funck, E. Pretsch, and W. Simon, Heir. Chim. Acta 60, 2326 (1977). 13 In part reproduced from W. Simon, D. Ammann, M. Oehme, and W. E. Morf, Ann. N. E Acad. Sci. 307, 52 (1978).
(ETH 1001)
~o
o
J N ~ o ~
O (ETH 1097)
~°
O
~
~o O
@o~.~~ o
(ETH149)
~°
O
I
~]~O f
~
o
N
(ETH 227) ~
(ETH lST)
O
~°
Fla. I. Structure of the ionophores ETH 1001, ETH 129, ETH 1097, ETH 149, ETH 227,
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
443
emf
REFERENCE HALF-CELL
INNER REFERENCE HALF-CELL
FILLING SOLUTION
REFERENCE ELECTROLYTE
"'S
BODY
ELECTRODE
~!iSOLUT~i
ION SELECTIVE MEMBRANE
~i~/i!~ ION SELECTIVE ELECTRODE
~,,i
ELECTRODE
BODY
--DIAPHRAGM
REFERENCE ELECTRODE
FIG. 2. Schematic diagram of a membrane electrode circuit and cell assembly.
Ca 2+, Eq. (1) reduces to Eca = E~a + s
log(aca)
(2)
For an electrode with an ideal Ca 2÷ response, Eq. (2) corresponds to the Nernst equation where the slope s is given by s = 2.303 RT/2F = 29.58 mV (25 ° C)
(3)
From this the basic response toward Ca 2+ is derived as 1 mV = 8% change in a ca
(4)
In practice, slopes of the electrode response above and below 29.6 mV are observed. One of the most important criteria to characterize an electrode is its selectivity toward possible interfering ions. This may be described by specifying selectivity factors K cPot ~ . For systems with the primary ion Ca 2÷ and only one interfering Ion M z+, these selectivity parameters are defined by the response function E = E~a + s log [aca + ,t"P°tCaM~,,M~2/~1j jt~
(la)
Different methods have been proposed for the evaluation of selectivity factors. In the separate solution method the potential of a cell assembly (Fig. 2) is measured with each of two separate solutions, one containing Ca z+ at the activity aca (but no M~+), the other containing the ion M ~+ at the activity aM (but no Ca~÷). The first measured value Eca is given by Eq.
444
SPECIALIZED TECHNIQUES
[40]
(2), whereas the second potential EM is described by o Pot 21z E It = Eca+ s log [Kc~(aM) ]
(5)
Finally, K crot ~ may be determined as follows KPot
CaM
=
IO(EM_Eca)/S
ac____g._~ (aM)2/z
(6)
In the fixed interference method the cell potential is measured on solutions of constant level of interference, aM, and varying activity of Ca 2+. The observed potential values E are plotted versus the logarithm of the calcium activity (identical to -pCa). The intersection of the extrapolation of the linear portions of this curve, corresponding to Ec~ and EM, respectively, indicates the value aca, which is to be used to calculate gca rotM. At the intersection point E c a = EM holds and therefore K cPot~
--
(7)
aca (a~)2/z
Some selectivities are given in Table I. Due to the rather different methods of assessment of K crot ~ values, there are substantial discrepancies for similar and even for identical membranes.13 The detection limits of ion selective electrodes are mainly determined by contaminations of the sample solution stemming from impurities in the reagents, leaching of ions out of the membrane, or intentionally introduced background electrolytes. By using Ca2+-buffered solutions, the contamination of the sample solution by Ca z+ (e.g., from the water used or from containers) is eliminated, and therefore the detection limit is ideally lowered and is mainly given by the possible interfering ions. Similarly the determination of selectivity factors leads to smaller values of K~°tatatata~if TABLE I SELECTIVITIES, log KCaM,P°tOBTAINED USING NEUTRAL CARRIER MEMBRANE ELECTRODES (PHILIPS IS-561 Ca2+) a M,+ = CaZ+ 0
Na +
K+
Mg2+
SrZ+
Ba2+
H+
Zn2+
-5.00 b -6.10 a
-5.22 b
-5.10 b
-2.09 c
-3.23 ~
-0.05 b -4.39 ~
-3.85 ~
a See Simon e t a l . 13 M e m b r a n e b a s e d on ligand E T H 1001 (PVC, o - n i t r o p h e n y l oc t yl ether as m e m b r a n e solvent). b F i x e d interference method, using no Ca2+-buffers. c Sep arate solution method. a F i x e d interference method, using CaZ+-buffered solutions.
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
445
Ca2+-buffered solutions are used. In the absence of metal buffers, the selectivity factors often reflect the quality of the reagents used and may not be representative of the electrode characteristics. The lowest detection limits documented so far are 5 × 10-9 M Ca 2÷ and 6 x 10-7 M Ca z+ for a neutral carrier macroelectrode in Ca2+-buffered (10 -~ M Na + present) and unbuffered (0.333 M Mg2÷ present) systems, respectively. Comparable values are reported for Ca 2÷ microelectrodes. ~a Ion Selective Electrodes in Biochemical Systems. In most biological studies it is important to distinguish between total concentrations and free ions. This is particularly important for the metals of class IIA, and especially for Ca z+, which very easily forms complexes with a variety of biological ligands. Indeed, in most extra- and intracellular fluids, only between 1 and 2% of the total Ca 2+ is present as a free ion. ~4-~6 A variety of methods can measure directly total Ca 2÷ concentrations in appropriately treated biological samples (e.g., emission spectroscopy, atomic absorption spectroscopy). However, the " f r e e " Ca 2÷ concentration, particularly inside cells, is often the most important variable. This can be measured directly with the photoprotein aequorin, ar'~8 which luminesces blue light (h = 470 nm) in the presence of Ca '+. The protein can be microinjected into cells, thus affording a continuous readout of the intracellular ionic Ca 2+. The method is highly sensitive (detection limit at least 10-7 M). However, other cations such as lanthanides, and those of Group IIA (except Mg2÷) also evoke aequorin luminescence, x9 In addition, high concentrations of Na ÷ and/or K + have an inhibitory effect.29-22 The other methods employed so far for the determination of free Ca z+ concentrations at the low levels present in most biological materials are indirect and have several drawbacks. They involve the use of a series of metallochromic indicators, having various detection limits (e.g., murexide "s about 10-n M, arsenazo 11I24 about 10-8 M). The chief disad14A. L. Hodgkin and R. P. Keynes, J. Physiol. (London) 138, 253 (1957). 15p. F. Baker, A. L. Hodgkin, and E. B. Ridgway, J. Physiol. (London) 218, 709 (1971). 16E. Carafoli and M. Crompton, Curr. Top. Mere. Transp. 10, 151 (1978). lr O. Shimomura, F. H, Johnson, and Y. Saiga, Science 140, 1339 (1963). 18O. Shimomura, F. H. Johnson, and Y. Saiga, J. Cell. Comp. Physiol. 59, 223 (1962). 19j. R. Blinks, F. G. Prendergast, and D. G. Allen, Pharmacol. Rev. 28, 1 (1976). 2o F. G. Prendergast, D. G. Allen, and J. R. Blinks, in "Calcium Binding Proteins and Calcium Function" (R. H. Wasserman et al., eds.), p. 469. North-Holland Publ., Amsterdam, 1977. zl D. G. Moisescu, C. C. Ashley, and A. K. Campbell, Biochim. Biophys. Acta 396, 133 (1975). z2 D. G. Moisescu and C. C. Ashley, Biochim. Biophys. Acta 460, 189 (1977). z3 A. Scarpa, Vol. 24, p. 343. 24 R. Di Polo, J. Requena, F. J. Brinley, L. J. Mullins, A. Scarpa, and T. Tiffert, J. Gen. Physiol. 67, 433 (1976).
446
SPECIALIZED TECHNIQUES
[40]
vantages of these methods are the interference from most divalent and trivalent cations, and also some instability of the reagent color. An additional disadvantage in kinetically measuring the transport of Ca z+ across membranes is the possible penetration of the metallochromic indicator through the membrane. 2s Calcium electrodes have none of the disadvantages of the indirect metallochromic methods (see Crompton and Carafoli [28], this volume). As mentioned above, they measure free Ca z+ with optimal selectivity and adequate response time. At the present stage, their detection limits in biological samples, in the absence of Ca 2+ buffers, are not as low as those of the most sensitive metallochromic indicators. However, in Ca 2÷ buffered biological samples, in the presence of a background NaCI concentration of 0.1 M, the detection limit of Ca z÷ microelectrodes has been found to lie in the range of 10-r-10 -s M. One additional advantage of the Ca 2+ electrodes is indeed the possibility of miniaturization, which can afford the means to obtain a continuous readout of the free Ca z+ ion activity inside cells. Microelectrodes based on ligand ETH 1001 are obtained by interposing the ion-selective liquid within a glass capillary z° (tip diameter around 1 /zm, see also section on ion selective electrodes, general) between the sample solution and the internal filling solution (Fig. 2). Their use in the measurement of ion activities, also in the intracellular ambient, is in full progress (see Nicholson et al.). zr-29a Use of Neutral Ionophores of Measure Transmembrane Electrical Potentials. In moving cations across the biological membrane the positively charged neutral ionophore-cation complexes may respond to a preexisting transmembrane electrical potential (electrodialytic movement) or they may themselves generate an electrical potential, as migration occurs in 25 L. Blayway, H. Thomas, J. Muir, and A. Henderson, Biochim. Biophys. Acta 470, 128 (1977). 2e M. Oehme, M. Kessler, and W. Simon, Chimia 30, 204 (1976). zrC. Nicholson, R. Steinberg, H. St6ckle, and G. Ten Bruggencate, Neurosci. Lett. 3, 315 (1975). 2s C. Nicholson, G. Ten Bruggencate, R. Steinberg, and H. St6ckle, Proc. Natl. Acad. Sci. U.S.A. 74, 1287 (1977). ~9 U. Heinemann, H. D. Lux, and M. J. Gutnick, Exp. Brain Res, 27, 237 (1977). ~ga S o m e substances commonly used in biochemical systems have detrimental effectson the P V C Ca~+-selective electrodes, and should be avoided or used under carefully controlled conditions. A m o n g them are detergents (SDS, Lubrol, Triton X-100, etc.),proton-carrying compounds (e.g.,uncouplers of oxidative phosphorylation), and ionophores having various selectivities.Underestimalions of the free Ca 2+ concentration can of course be induced by compounds able to buffer Ca 2+ in the system (e.g.,inorganic phosphate, adenine nucleotide, citricacid, etc.).To avoid drifts,and to minimize noise, care should be taken to buffer the p H of the medium well, to keep its temperature constant, and to provide a reasonable ionic strength.
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
447
response to a concentration gradient of the cation (electrogenic translocation). In either case, the neutral ionophore makes possible the transfer of charge across the biological membrane. After the charged complex has migrated across the biological membrane, the uncharged (free) ionophore travels back to the original membrane side to complex the cation again and repeat the cycle of translocation. Making some assumptions (see below), the equilibrium expression for the activity of the cation transported is given by the Nernst equation RT
AE = ~
[C+]l
In [C+]2
(8)
where [C+]l and [C+]2 are the activities of the transported cation in the two compartments separated by the membrane. Neutral ionophores can thus be used to measure electrical potentials (AE) across biological membranes, based on measurements of the equilibrium distribution of the ionophore selective cation. These measurements obviously require that activities and not concentrations be measured, which may be very difficult in subceUular organelles such as the mitochondria. In these cases, the activities of the cation in the intraorganellar space must be evaluated by indirect, and often unreliable, means. Alternatively, the percentage of "bound" (osmotically inactive) cation, can be reduced by allowing the penetration into the organelle of anions which form soluble salts with the cation studied. Another possible complication in determining membrane potentials using neutral ionophore-cation complexes, is the possible existence of natural cation pumping activities, which could pump out (part of) the cation transported, thus causing a deviation from the Nernstian equilibrium. Whenever possible, "artificial" cations, not likely to be substrates for natural pumps, should thus be used to form charged complexes with the ionophore. It must also be stressed that the discussion above is based on the assumption that (a) the neutral ionophore does not interact with other ions present in the system that could influence the transmembrane electrical potential and (b) the transmembrane electrical potential is not influenced by the movement of the transported ion, i.e., it is continually regenerated. Determinations of electrical potential differences across various biological membranes have been repeatedly performed using neutral ionophores (e.g., valinomycin) and monovalent cations such as K + or Rb+. 3°-32 In this case, the measurements are relatively simple, since for ao p. Mitchell and J. Moyle, Eur. J. Biochem. 7, 471 (1%9). 3~D. G. Nicholls, Eur. J. Biochem. 50, 305 (1974). 3z E. Padan and H. Rottenberg, Eur. J. Biochern. 40, 431 (1973).
448
SPECIALIZED TECHNIQUES
[41]
these monovalent cations the total and free concentrations essentially coincide. An additional advantage, in these cases of monovalent cations, is that their concentration gradients across most biological membranes are in the range 10-1 to 10-z M. This, together with the fact that smaller equilibrium gradients (z -- 1) than for divalent cations are predicted, facilitates the measurements. Indeed, the concentrations of monovalent cations in membrane-separated compartments never drop below the levels which afford reasonably secure measurements. In the case of a divalent cation such as Ca 2÷, the situation is entirely different. Its ionic concentration in most membrane-separated biological compartments is very low, and in addition, for equal membrane potentials, bigger gradients than in the case of monovalent cations are expected (z = 2). To take the example of the mitochondria, where the membrane potential maintained by respiration is about 180 mV, at equilibrium one would expect, for a charge number of 2, an in/out Ca .'÷ activity ratio of 10-". Considering that the free Ca .'÷ ion activity in the intramitochondrial water is not more than 10-3 M, 38 one would expect an equilibrium extramitochondrial Ca .'÷ activity value of not more than 10-gM, which is far too low to be measured by any of the methods presently available. In attempting to correlate the free Ca .,÷ distribution with the transmembrane electrical potential, one must therefore accept a compromise, i.e., decrease artificially the latter in a controlled way to reduce the transmembrane gradient of free Ca ~÷ to levels where the extramitochondrial cation can be safely measured. This can be achieved by the controlled addition to the system of protoncarrying uncouplers of oxidative phosphorylation. 33,34 33 M. Crompton and I. Heid, Eur. J. Biochem. in press (1978). 34 H. Rottenberg and A. Scarpa, Biochemistry 13, 4811 (1974).
[41] T h e H y d r o g e n P e r o x i d e S e n s i n g P l a t i n u m A n o d e as a n Analytical Enzyme Electrode
By
L E L A N D C . C L A R K , JR.
Introduction
General Rationale. Oxidases are oxidoreductases where oxygen is the acceptor of the hydrogen donated by the substrate. While oxidases form only a fraction of the enzymes known today and, in fact, only a small fraction of the known oxidoreductases, they are important in bioanalytical chemistry because they generate a substance, hydrogen peroxide, which can be measured electrochemically. METHODS IN ENZYMOLOGY,VOL. LVI
Copyright © 1979by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181956-6
D E S I G N , PROPERTIES, A N D A P P L I C A T I O N S
439
[40] D e s i g n , P r o p e r t i e s , a n d A p p l i c a t i o n s o f N e u t r a l Ionophores By WILHELM SIMON and ERNESTO CARAFOLI Synthetic Neutral Ionophores Design Features. Electrically neutral, lipophilic ion-complexing agents of rather small relative molar mass are known to behave as ionophores or ion carriers. 1 They have the capability to selectively extract ions from aqueous solutions into a hydrophobic membrane phase and to transport these ions across such barriers by carrier translocation. In order for a ligand to behave as an ionophore for cations, several aspects have to be considered (see also Simon et al.2). a. A carrier molecule should be composed of polar and nonpolar groups. b. The carder should be able to assume a stable conformation that provides a cavity, surrounded by the polar groups, suitable for the uptake of a cation, while the nonpolar groups form a lipophilic shell around the coordination sphere. These groups must ensure sufficiently large lipid solubility for ligand and complex. This is one reason why classic electrically charged complexing agents, such as ethylenediamine tetraacetic acid (EDTA), will not behave as carders in membrane systems. c. Among the polar groups of the ligand sphere, there should be preferably five to eight, but not more than twelve coordinating sites, such as oxygen atoms. d. High selectivities are achieved by locking the coordinating sites into a rigid arrangement around the cavity. Such rigidity can be enhanced by the presence of bridged structures or hydrogen bonds. Within one group of the periodic system, the cation that best fits into the offered cavity is preferred. Ideally, all cations should be forced into accepting the same given number of coordinating groups. e. Notwithstanding requirement (d), the ligand should be flexible enough to allow a sufficiently fast ion exchange. This is possible only with a stepwise substitution of the solvent molecules by the ligand groups. Thus, a compromise between stability (d) and exchange rate (e) has to be found. 1Yu. A. Ovchinnikov, V. T. Ivanov, and A. M. Shkrob, BBA Libr. 12, 1 (1974). 2 W. Simon, W. E. Morf, and D. Ammann, in "'Calcium Binding Proteins and Calcium Function" (R. H. Wasserman et al., eds.), p. 50. North-Holland Publ., Amsterdam, 1977.
METHODS IN ENZYMOLOGY,VOL. LVI
Copyright © 1979by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181956-6
440
SPECIALIZED TECHNIQUES
[40]
f. To guarantee an adequate mobility, the overall dimensions of a carrier should be rather small but still compatible with high lipid solubility. Attractive binding sites are ligand atoms which are capable of competing with water molecules in the complexation of ions. Through variation of molecular parameters, such as dipole moment and polarizability of the binding sites as well as the van der Waals radius of the ligand atoms, the interaction selectivity may be considerably influenced (for details, see Simon et al.2"3). It is a well known fact that complexes of polydentate ligands have enhanced stability over their unidentate counterparts (chelate effect), and complexes of macrocyclic ligands are, as a rule, more stable than those of noncyclic polydentate ligands (macrocyclic effect). 3,4 According to Adamson,5 the chelate effect is largely a consequence of the asymmetry of the standard reference state. Stabilization effects beyond what has been discussed may be due to a reduction in translational and/or rotational entropy of the free polydentate ligand. In macrocyclic and especially in macropolycyclic ligands, repulsions, e.g. between binding sites, are already built in and, in addition, optimal solvation of the free ligand sites may be prevented so that the formation of the complexes is further favored.4 Due to favoured conformations of a multidentate ligand of a given constitution, the coordination shell (i.e., coordination number and cavity radius) may be predetermined and therefore selectivity can be induced even by non-macrocyclic molecules. Indeed, the non-macrocyclic neutral carriers shown in Fig. 1 are capable of inducing extremely high selectivity for cations in certain membranes. 6 The ligands ETH 1001r (Fluka AG, Buchs, Switzerland) and ETH 1293 exhibit rather high selectivity for Ca z÷. ETH 1001 and ETH 129 may form 1:2 and 1:3 Ca2+-ligand complexes, respectively. Since the ether and amide oxygen atoms participate in the coordination of the calcium ion, there will be a coordination sphere of 8 oxygen atoms for ETH 1001 and of 9 oxygen atoms for ETH 129. In both cases the oxygen-Ca 2÷ distance is a W. E. Moff, R. Bissig, D. Ammann, E. Pretsch, and W. Simmon, in "Multidentate Macrocyclic Molecules" (J. J. Christensen and R. M. Izatt, eds.). Wiley (Interscience), New York (in press). 4 J.-M. Lehn, Struct. Bonding (Berlin), 16, 1 (1973). 5 A. W. Adamson, J. A m . Chem. Soc. 76, 1578 (1954). D. Ammann, R. Bissig, Z. Cimerman, U. Fiedler, M. Giiggi, W. E. Morf, M. Oehme, H. Osswald, E. Pretsch, and W. Simon, in "Ion and Enzyme Electrodes in Biology and Medicine" (M. Kessler, et al., eds.), p. 22. Urban & Schwarzenberg, Munich, 1976. 7 D. Ammann, M. Giiggi, E. Pretsch, and W. Simon, Anal. Lett. 8, 709 (1975).
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
441
around 2.4 A, which roughly corresponds to the sum of the radii of oxygen and Ca2+.3'8'9 The synthetic ionophores ETH 1097,1° ETH 227,11 and ETH 1576 are capable of inducing selectivity for Na ÷ over K ÷ whereas ETH 149TM has interesting selectivity for Li+. 6,1z Applications of Neutral Ionophores
Ion Selective Electrodes, General.13 The ionophores shown in Fig. 1 may be used as components in liquid membrand electrodes for the measurement of ion activities. 6 Ideally, such an electrode cell assembly (Fig. 2) generates an electric potential difference or emf that is proportional to the logarithm of the activity of the ion to be measured (Nernstian response). In the classic arrangement, the ion selective membrane consisted of a filter paper impregnated with a water-immiscible solution of the ion exchanger. Significant, especially mechanical, improvements were obtained by incorporating the electroactive material into polyvinylchloride (PVC). For the preparation of microelectrodes with tip diameters of around 1/zm, the ion selective liquid may be placed directly into a capillary between the sample solution and the internal filling solution (see Fig. 2). Because of the extremely high selectivity of the Ca2+-ionophore ETH 1001,13 and the interest in the measurement of Ca 2+ in biological systems, we will focus here on the determination of the activity aca using ion selective electrodes (for further details, see Simon et al. 13). A semiempirical and fairly successful approach, which has been recommended by the IUPAC, to treat real membrane electrode systems is offered by the extended Nicolsky-Eisenman equation: E
g~a +s
log (aca "~ ~ g~aatM (aM) 2tz" /)
(1)
M
where E is the cell potential (emf), E~a the reference potential at constant temperature; s the slope of electrode response function, aca the activity of primary ion Ca 2+ in sample solution, a~ the activity of an interfering ion M (charge zM) in sample solution, g cPot a M the potentiometric selectivity coefficient in respect to ion M. If there is no interference by ions other than 8 K. Neupert-Laves and M. Dobler, Helv. Chim. Acta 60, 1861 (1977) M. Dobler, private communication. 10 D. Ammann, E. Pretsch, and W. Simon, Anal. Lett. 7, 23 (1974). 11M. Giiggi, M. Oehme, E. Pretsch, and W. Simon, Helv. Chim. Acta 59, 2417 (1976). 12N. N. L, Kirsch, R. J. Funck, E. Pretsch, and W. Simon, Heir. Chim. Acta 60, 2326 (1977). 13 In part reproduced from W. Simon, D. Ammann, M. Oehme, and W. E. Morf, Ann. N. E Acad. Sci. 307, 52 (1978).
(ETH 1001)
~o
o
J N ~ o ~
O (ETH 1097)
~°
O
~
~o O
@o~.~~ o
(ETH149)
~°
O
I
~]~O f
~
o
N
(ETH 227) ~
(ETH lST)
O
~°
Fla. I. Structure of the ionophores ETH 1001, ETH 129, ETH 1097, ETH 149, ETH 227,
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
443
emf
REFERENCE HALF-CELL
INNER REFERENCE HALF-CELL
FILLING SOLUTION
REFERENCE ELECTROLYTE
"'S
BODY
ELECTRODE
~!iSOLUT~i
ION SELECTIVE MEMBRANE
~i~/i!~ ION SELECTIVE ELECTRODE
~,,i
ELECTRODE
BODY
--DIAPHRAGM
REFERENCE ELECTRODE
FIG. 2. Schematic diagram of a membrane electrode circuit and cell assembly.
Ca 2+, Eq. (1) reduces to Eca = E~a + s
log(aca)
(2)
For an electrode with an ideal Ca 2÷ response, Eq. (2) corresponds to the Nernst equation where the slope s is given by s = 2.303 RT/2F = 29.58 mV (25 ° C)
(3)
From this the basic response toward Ca 2+ is derived as 1 mV = 8% change in a ca
(4)
In practice, slopes of the electrode response above and below 29.6 mV are observed. One of the most important criteria to characterize an electrode is its selectivity toward possible interfering ions. This may be described by specifying selectivity factors K cPot ~ . For systems with the primary ion Ca 2÷ and only one interfering Ion M z+, these selectivity parameters are defined by the response function E = E~a + s log [aca + ,t"P°tCaM~,,M~2/~1j jt~
(la)
Different methods have been proposed for the evaluation of selectivity factors. In the separate solution method the potential of a cell assembly (Fig. 2) is measured with each of two separate solutions, one containing Ca z+ at the activity aca (but no M~+), the other containing the ion M ~+ at the activity aM (but no Ca~÷). The first measured value Eca is given by Eq.
444
SPECIALIZED TECHNIQUES
[40]
(2), whereas the second potential EM is described by o Pot 21z E It = Eca+ s log [Kc~(aM) ]
(5)
Finally, K crot ~ may be determined as follows KPot
CaM
=
IO(EM_Eca)/S
ac____g._~ (aM)2/z
(6)
In the fixed interference method the cell potential is measured on solutions of constant level of interference, aM, and varying activity of Ca 2+. The observed potential values E are plotted versus the logarithm of the calcium activity (identical to -pCa). The intersection of the extrapolation of the linear portions of this curve, corresponding to Ec~ and EM, respectively, indicates the value aca, which is to be used to calculate gca rotM. At the intersection point E c a = EM holds and therefore K cPot~
--
(7)
aca (a~)2/z
Some selectivities are given in Table I. Due to the rather different methods of assessment of K crot ~ values, there are substantial discrepancies for similar and even for identical membranes.13 The detection limits of ion selective electrodes are mainly determined by contaminations of the sample solution stemming from impurities in the reagents, leaching of ions out of the membrane, or intentionally introduced background electrolytes. By using Ca2+-buffered solutions, the contamination of the sample solution by Ca z+ (e.g., from the water used or from containers) is eliminated, and therefore the detection limit is ideally lowered and is mainly given by the possible interfering ions. Similarly the determination of selectivity factors leads to smaller values of K~°tatatata~if TABLE I SELECTIVITIES, log KCaM,P°tOBTAINED USING NEUTRAL CARRIER MEMBRANE ELECTRODES (PHILIPS IS-561 Ca2+) a M,+ = CaZ+ 0
Na +
K+
Mg2+
SrZ+
Ba2+
H+
Zn2+
-5.00 b -6.10 a
-5.22 b
-5.10 b
-2.09 c
-3.23 ~
-0.05 b -4.39 ~
-3.85 ~
a See Simon e t a l . 13 M e m b r a n e b a s e d on ligand E T H 1001 (PVC, o - n i t r o p h e n y l oc t yl ether as m e m b r a n e solvent). b F i x e d interference method, using no Ca2+-buffers. c Sep arate solution method. a F i x e d interference method, using CaZ+-buffered solutions.
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
445
Ca2+-buffered solutions are used. In the absence of metal buffers, the selectivity factors often reflect the quality of the reagents used and may not be representative of the electrode characteristics. The lowest detection limits documented so far are 5 × 10-9 M Ca 2÷ and 6 x 10-7 M Ca z+ for a neutral carrier macroelectrode in Ca2+-buffered (10 -~ M Na + present) and unbuffered (0.333 M Mg2÷ present) systems, respectively. Comparable values are reported for Ca 2÷ microelectrodes. ~a Ion Selective Electrodes in Biochemical Systems. In most biological studies it is important to distinguish between total concentrations and free ions. This is particularly important for the metals of class IIA, and especially for Ca z+, which very easily forms complexes with a variety of biological ligands. Indeed, in most extra- and intracellular fluids, only between 1 and 2% of the total Ca 2+ is present as a free ion. ~4-~6 A variety of methods can measure directly total Ca 2÷ concentrations in appropriately treated biological samples (e.g., emission spectroscopy, atomic absorption spectroscopy). However, the " f r e e " Ca 2÷ concentration, particularly inside cells, is often the most important variable. This can be measured directly with the photoprotein aequorin, ar'~8 which luminesces blue light (h = 470 nm) in the presence of Ca '+. The protein can be microinjected into cells, thus affording a continuous readout of the intracellular ionic Ca 2+. The method is highly sensitive (detection limit at least 10-7 M). However, other cations such as lanthanides, and those of Group IIA (except Mg2÷) also evoke aequorin luminescence, x9 In addition, high concentrations of Na ÷ and/or K + have an inhibitory effect.29-22 The other methods employed so far for the determination of free Ca z+ concentrations at the low levels present in most biological materials are indirect and have several drawbacks. They involve the use of a series of metallochromic indicators, having various detection limits (e.g., murexide "s about 10-n M, arsenazo 11I24 about 10-8 M). The chief disad14A. L. Hodgkin and R. P. Keynes, J. Physiol. (London) 138, 253 (1957). 15p. F. Baker, A. L. Hodgkin, and E. B. Ridgway, J. Physiol. (London) 218, 709 (1971). 16E. Carafoli and M. Crompton, Curr. Top. Mere. Transp. 10, 151 (1978). lr O. Shimomura, F. H, Johnson, and Y. Saiga, Science 140, 1339 (1963). 18O. Shimomura, F. H. Johnson, and Y. Saiga, J. Cell. Comp. Physiol. 59, 223 (1962). 19j. R. Blinks, F. G. Prendergast, and D. G. Allen, Pharmacol. Rev. 28, 1 (1976). 2o F. G. Prendergast, D. G. Allen, and J. R. Blinks, in "Calcium Binding Proteins and Calcium Function" (R. H. Wasserman et al., eds.), p. 469. North-Holland Publ., Amsterdam, 1977. zl D. G. Moisescu, C. C. Ashley, and A. K. Campbell, Biochim. Biophys. Acta 396, 133 (1975). z2 D. G. Moisescu and C. C. Ashley, Biochim. Biophys. Acta 460, 189 (1977). z3 A. Scarpa, Vol. 24, p. 343. 24 R. Di Polo, J. Requena, F. J. Brinley, L. J. Mullins, A. Scarpa, and T. Tiffert, J. Gen. Physiol. 67, 433 (1976).
446
SPECIALIZED TECHNIQUES
[40]
vantages of these methods are the interference from most divalent and trivalent cations, and also some instability of the reagent color. An additional disadvantage in kinetically measuring the transport of Ca z+ across membranes is the possible penetration of the metallochromic indicator through the membrane. 2s Calcium electrodes have none of the disadvantages of the indirect metallochromic methods (see Crompton and Carafoli [28], this volume). As mentioned above, they measure free Ca z+ with optimal selectivity and adequate response time. At the present stage, their detection limits in biological samples, in the absence of Ca 2+ buffers, are not as low as those of the most sensitive metallochromic indicators. However, in Ca 2÷ buffered biological samples, in the presence of a background NaCI concentration of 0.1 M, the detection limit of Ca z÷ microelectrodes has been found to lie in the range of 10-r-10 -s M. One additional advantage of the Ca 2+ electrodes is indeed the possibility of miniaturization, which can afford the means to obtain a continuous readout of the free Ca z+ ion activity inside cells. Microelectrodes based on ligand ETH 1001 are obtained by interposing the ion-selective liquid within a glass capillary z° (tip diameter around 1 /zm, see also section on ion selective electrodes, general) between the sample solution and the internal filling solution (Fig. 2). Their use in the measurement of ion activities, also in the intracellular ambient, is in full progress (see Nicholson et al.). zr-29a Use of Neutral Ionophores of Measure Transmembrane Electrical Potentials. In moving cations across the biological membrane the positively charged neutral ionophore-cation complexes may respond to a preexisting transmembrane electrical potential (electrodialytic movement) or they may themselves generate an electrical potential, as migration occurs in 25 L. Blayway, H. Thomas, J. Muir, and A. Henderson, Biochim. Biophys. Acta 470, 128 (1977). 2e M. Oehme, M. Kessler, and W. Simon, Chimia 30, 204 (1976). zrC. Nicholson, R. Steinberg, H. St6ckle, and G. Ten Bruggencate, Neurosci. Lett. 3, 315 (1975). 2s C. Nicholson, G. Ten Bruggencate, R. Steinberg, and H. St6ckle, Proc. Natl. Acad. Sci. U.S.A. 74, 1287 (1977). ~9 U. Heinemann, H. D. Lux, and M. J. Gutnick, Exp. Brain Res, 27, 237 (1977). ~ga S o m e substances commonly used in biochemical systems have detrimental effectson the P V C Ca~+-selective electrodes, and should be avoided or used under carefully controlled conditions. A m o n g them are detergents (SDS, Lubrol, Triton X-100, etc.),proton-carrying compounds (e.g.,uncouplers of oxidative phosphorylation), and ionophores having various selectivities.Underestimalions of the free Ca 2+ concentration can of course be induced by compounds able to buffer Ca 2+ in the system (e.g.,inorganic phosphate, adenine nucleotide, citricacid, etc.).To avoid drifts,and to minimize noise, care should be taken to buffer the p H of the medium well, to keep its temperature constant, and to provide a reasonable ionic strength.
[40]
DESIGN, PROPERTIES, AND APPLICATIONS
447
response to a concentration gradient of the cation (electrogenic translocation). In either case, the neutral ionophore makes possible the transfer of charge across the biological membrane. After the charged complex has migrated across the biological membrane, the uncharged (free) ionophore travels back to the original membrane side to complex the cation again and repeat the cycle of translocation. Making some assumptions (see below), the equilibrium expression for the activity of the cation transported is given by the Nernst equation RT
AE = ~
[C+]l
In [C+]2
(8)
where [C+]l and [C+]2 are the activities of the transported cation in the two compartments separated by the membrane. Neutral ionophores can thus be used to measure electrical potentials (AE) across biological membranes, based on measurements of the equilibrium distribution of the ionophore selective cation. These measurements obviously require that activities and not concentrations be measured, which may be very difficult in subceUular organelles such as the mitochondria. In these cases, the activities of the cation in the intraorganellar space must be evaluated by indirect, and often unreliable, means. Alternatively, the percentage of "bound" (osmotically inactive) cation, can be reduced by allowing the penetration into the organelle of anions which form soluble salts with the cation studied. Another possible complication in determining membrane potentials using neutral ionophore-cation complexes, is the possible existence of natural cation pumping activities, which could pump out (part of) the cation transported, thus causing a deviation from the Nernstian equilibrium. Whenever possible, "artificial" cations, not likely to be substrates for natural pumps, should thus be used to form charged complexes with the ionophore. It must also be stressed that the discussion above is based on the assumption that (a) the neutral ionophore does not interact with other ions present in the system that could influence the transmembrane electrical potential and (b) the transmembrane electrical potential is not influenced by the movement of the transported ion, i.e., it is continually regenerated. Determinations of electrical potential differences across various biological membranes have been repeatedly performed using neutral ionophores (e.g., valinomycin) and monovalent cations such as K + or Rb+. 3°-32 In this case, the measurements are relatively simple, since for ao p. Mitchell and J. Moyle, Eur. J. Biochem. 7, 471 (1%9). 3~D. G. Nicholls, Eur. J. Biochem. 50, 305 (1974). 3z E. Padan and H. Rottenberg, Eur. J. Biochern. 40, 431 (1973).
448
SPECIALIZED TECHNIQUES
[41]
these monovalent cations the total and free concentrations essentially coincide. An additional advantage, in these cases of monovalent cations, is that their concentration gradients across most biological membranes are in the range 10-1 to 10-z M. This, together with the fact that smaller equilibrium gradients (z -- 1) than for divalent cations are predicted, facilitates the measurements. Indeed, the concentrations of monovalent cations in membrane-separated compartments never drop below the levels which afford reasonably secure measurements. In the case of a divalent cation such as Ca 2÷, the situation is entirely different. Its ionic concentration in most membrane-separated biological compartments is very low, and in addition, for equal membrane potentials, bigger gradients than in the case of monovalent cations are expected (z = 2). To take the example of the mitochondria, where the membrane potential maintained by respiration is about 180 mV, at equilibrium one would expect, for a charge number of 2, an in/out Ca .'÷ activity ratio of 10-". Considering that the free Ca .'÷ ion activity in the intramitochondrial water is not more than 10-3 M, 38 one would expect an equilibrium extramitochondrial Ca .'÷ activity value of not more than 10-gM, which is far too low to be measured by any of the methods presently available. In attempting to correlate the free Ca .,÷ distribution with the transmembrane electrical potential, one must therefore accept a compromise, i.e., decrease artificially the latter in a controlled way to reduce the transmembrane gradient of free Ca ~÷ to levels where the extramitochondrial cation can be safely measured. This can be achieved by the controlled addition to the system of protoncarrying uncouplers of oxidative phosphorylation. 33,34 33 M. Crompton and I. Heid, Eur. J. Biochem. in press (1978). 34 H. Rottenberg and A. Scarpa, Biochemistry 13, 4811 (1974).
[41] T h e H y d r o g e n P e r o x i d e S e n s i n g P l a t i n u m A n o d e as a n Analytical Enzyme Electrode
By
L E L A N D C . C L A R K , JR.
Introduction
General Rationale. Oxidases are oxidoreductases where oxygen is the acceptor of the hydrogen donated by the substrate. While oxidases form only a fraction of the enzymes known today and, in fact, only a small fraction of the known oxidoreductases, they are important in bioanalytical chemistry because they generate a substance, hydrogen peroxide, which can be measured electrochemically. METHODS IN ENZYMOLOGY,VOL. LVI
Copyright © 1979by Academic Press, Inc. All rightsof reproduction in any form reserved. ISBN 0-12-181956-6
