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Application of an SH surface acoustic wave to the analysis of catalysis on immobilized galactose oxidase
Journal of Molecular Catalysis, 74 (1992) 459-464 M2936
459
Application of an SH surface acoustic wave to the analysis of catalysis on immobilized galactose oxidase Yasunobu Inoue" and Yoshihiro Kato Department of Chemistry and Analysis Center, Nagaoka University of Technology, Nagaoka, Niigata 940-21 (Japan)
Abstract The surface acoustic wave (SAW) method, combined with measurements of reaction rates, was employed to investigate the role of the metal ion in the catalysis of a metalloenzyme. A device generating a shear horizontal leaky SAW was fabricated on which galactose oxidase (GAOD) was immobilized. The removal of Cu ions from immobilized GAOD to produce an inactive apoenzyme and its reverse process were monitored by changes in the SAW frequency. From the response in the SAW frequency during the oxidation of galactose, the formation of an enzyme-substrate complex was confirmed for GAOD, whereas the apoenzyme was found to have no capability of coordinating galactose, which was responsible for deactivation. Zn and Co ions were also accommodated in the apoenzyme to almost the same extent as Cu ions, but had no capability of coordinating galactose. It is concluded that the Cu ions control the coordination of galactose.
Introduction In an attempt to develop a method for analyzing the behavior of catalytic reactions on solid surfaces, we have focused on the great promise shown by surface acoustic waves (SAWs). Since the resonant frequency of the SAW is very sensitive to changes in mass loaded on the propagation path of the wave [1, 2 J, it is possible to measure even a trace amount of adsorbed species on the surface. Furthermore, a fact that measurements of the SAW frequency can be made independently of measurements of reaction rates permits determination of the amounts of the adsorbed species during the catalytic reactions. Recently we showed the availability of a shear horizontal (SH) leaky SAW to wave propagation in liquid phases and successfully applied it to examine the reaction mechanism of glucose oxidation on the immobilized glucose oxidase [3 J. Galactose oxidase is a metalloenzyme involving Cu ions as an essential metal ion and is active for the oxidation of galactose to produce galactohexodialdose and hydrogen peroxide. The removal of the Cu ions converts the active enzyme to an apoenzyme that has no catalytic activity. As for the role of the Cu ions, at least three questions remain unsolved. First, there *Author to whom correspondence should be addressed.
0304-5102/92/$5.00
© 1992 - Elsevier Sequoia. All rights reserved
460
are two possibilities for deactivation caused by Cu elimination, from a mechanistic point of view. One is that the apoenzyme itself has no capability of coordinating galactose, such that there is no chance for the reaction to proceed further. The other is that the apoenzyme is able to coordinate the substrate but loses the activity of oxidizing the coordinated species. The second question is whether or not metal ions other than Cu ions can be accommodated in the apoenzyme, and the third question is, if the accommodation takes place, whether or not the resulting enzymes have the ability to coordinate galactose. Finding answers to these questions will lead to understanding of the role of the Cu ions in the catalytic functions of GAOD. For this purpose, in addition to measurements of the catalytic activity, we need to monitor directly the amounts of the substrate coordinated on immobilized metalloenzymes and apoenzymes during the catalytic reactions, but so far there have been few studies employing such an approach [4, 5]. In the present study, the shear horizontal leaky SAW method was applied to the system of the immobilized GAOD and the apoenzyme.
Experimental The SAW device was the same as previously used [3]. The input and output interdigital transducer (IDT) electrodes, having a 200 JLm periodicity [6, 7], were fabricated photolithographically on a 36°-rotatedY-cut single crystal of LiTa03. The center frequency was 20.95 MHz in air. The catalyst was prepared by immobilizing galactose oxidase (GAOD) (Sigma Chemical) at pH = 7 in phosphate buffer solutions on the SAW device surface functionalized by a silane coupling agent, 3-aminopropyltriethoxysilane, and then by glutalaldehyde. The resonant SAW frequency of the catalyst was amplified by a feedback path in an oscillator loop and was monitored by a frequency counter. The SAW frequency change was converted to mass units according to the equation previously derived [3]. The catalyst was mounted on a special reaction cell for the simultaneous measurement of the SAW frequency and the reaction rates. The reaction rates at 298 K were determined from the amount of hydrogen peroxide produced, which was measured with a threeelectrode system using a potentiostat. The anode potential of the Pt working electrode was kept at +0.6 V vs. SCE.
Results After the resonant SAW frequency of the GAOD immobilized catalyst reached a constant level in the phosphate buffer solution at pH 8, a solution of a chelating agent, N ,N-diethyl-dithiocarbamic acid sodium salt, was added at a concentration of 10 roM. As shown in Fig. 1, the SAW frequency increased gradually and attained a saturation level. The total change in frequency amounted to 65 Hz. The solution examined was replaced by a fresh buffer
461 (a)
1
20 HZ
N
:r:
:;-
Chelating
agent
I 0
2
3
tlmin (I)
(b)
(3)
1.2 N
:r:
-
«c
Cu ion
OB Gal3.ctose
_ 0.4
1
20 Hz
( (2)
0
0
4 t /min
6
0
1 t/min
2
Fig. 1. Changes in SAW frequency: (a) addition of the chelating agent to a GAOD system; (b) after (a), addition of copper sulfate aqueous solution. Fig. 2. Catalytic activity for the oxidation of galactose on three enzymes: (1) GAOD; (2) apoenzyme; (3) Cu-apoenzyme. Conditions: pH 6, in 0.1 M phosphate buffer solution.
solution, and then the aqueous solution of copper sulfate was introduced. The frequency attenuated monotonically until it reached a constant level with a decrease of 65 Hz. The increase in the SAW frequency caused by the addition of the chelating agent was compensated for by an equal decrease caused by the addition of Cu ion solutions. This reversible change reflects the removal of Cu ions by the chelating agent from GAOD to form an apoenzyme and the accommodation of Cu ions to form GAOD. (In order to differentiate it from as-immobilized, original GAOD, this GAOD is hereafter referred to as Gu-apoenzyme.) The catalytic activity for the oxidation of galactose was compared for the three immobilized enzymes, GAOD, the apoenzyme and the Gu-apoenzyme. As shown in Fig. 2, GAOD has a considerable activity, whereas there is no catalytic activity for the apoenzyme. For the Gu-apoenzyme, the activity was equal to up to 85% of that of the original GAOD. Figure 3 shows changes in the SAW frequency due to the coordination of galactose during the catalytic oxidation. When the galactose substrate was added at a concentration of 1 mM, the SAW frequency of the immobilized GAOD showed a rapid decrease and reached a saturation level of 53 Hz in a short time. On the other hand, a negligibly small frequency change occurred for the apoenzyme. The frequency shift in the Gu-apoenzyme was 46 Hz, which was 87% of the shift in the original GAOD. The ability of apoenzyme to accommodate metal ions other than Cu ions was examined for Zn and Go ions, whose ionic radii are close to that of Cu ions. Figure 4 shows changes in the SAW frequency when the immobilized apoenzyme was exposed to the aqueous solutions of cobalt chloride and
462
N
! :I:
Co ion
10Hz
! Galactose
N~-(2)
; 12~---------m
Zn ion
1
20 HZ
o t
5 Imin
10
o
2
4
t I min
6
Fig. 3. Response of the SAWfrequency upon the addition of galactose: (1) GAOD; (2) apoenzyme; (3) Cu-apoenzyme. Experimental conditions are the same as in Fig. 2. Fig. 4. Changes in the SAW frequency when immobilized apoenzyme is exposed to the solutions of Co and Zn ions.
TABLE 1
Properties of the apoenzyme accommodating various metal ions Metal ions
Amount of metal ions accommodated (atoms cm- z)
Maximum amount of galactose coordinated (molecules cm- z)
Catalytic activity
Cu z+
5.9x 10 15 5.7x 1015 6.1 X 10 15
2.3 x 10 15 0 0
active inactive inactive
Znz + Co z+
zinc chloride. Negative frequency shifts were observed, indicative of the formation of Co- and Zn-apoenzymes, respectively. There was no significant difference in the total frequency shifts between Co (63 Hz) and Zn (65 Hz) ions, and it should be noted that the magnitudes of the frequency changes were similar to that for Cu ion. When galactose was introduced into the Zn-apoenzyme system, there was little change in the SAW frequency. A negligibly small response of the SAW frequency upon the addition of galactose was also observed for the Co-apoenzyme. Table 1 summarizes the properties of the apoenzymes accommodating the various metal ions. Figure 5 shows the amounts of galactose adsorbed, [M], on the immobilized GAOD during the catalytic reaction as a function of the galactose concentration, [S]. At low concentrations, the value of [M] increased remarkably, followed by a less rapid increase, and attained an almost saturation level above 2 roM. A straight line was obtained when [M]-I was plotted against [S ]-1.
463
2
o
2
4
6
[5], Galactose I mM Fig. 5. Amount of galactose coordinated on immobilized GAOD as a function of galactose concentration. Conditions: pH 6, 0.1 M phosphate buffer solution.
Discussion The oxidation of galactose on immobilized GAOD has been examined mostly by measuring the amount of H 20 2 produced using electrochemical methods [8]; the behavior of the coordinated species was not well understood. The present SAW results clearly indicate that no coordination of galactose on the immobilized GAOD occurs in the absence of Cu metal ions. Thus it is logical to assume that lack of apoenzyme catalytic activity can be associated with loss of the capability to form an enzyme-substrate complex. As shown in Table 1, Cu, Zn and Co ions are all accommodated in the apoenzyme to a similar extent. This indicates that the metal-ion coordination sites of the apoenzyme have no specificity in the selection of these metal ions. On the other hand, only the Cu-apoenzyme showed activity for galactose coordination, in contrast to the absence of activity of the Zn- and Co-apoenzymes. From these results, it is concluded that the recognition of the substrate through coordination is essential for the functions of the metalloenzyme. Cu ions have a high potential to arrange the highly ordered structural conformation suitable for the coordination of galactose. In the previous study [3] using the SAW method for the oxidation of glucose on the immobilized glucose oxidase (GOD), the Michaelis constant was found to be in good agreement with the equilibrium constant of glucose coordination during the reaction, and thus it was shown that almost all the coordinated glucose molecules participated in the catalytic reaction and that the coordination sites homogeneously contributed to the active sites. The linear relationship between [M]- 1 and [S]- 1 observed here suggests that the oxidation of galactose on the immobilized GAOD proceeds according to the Michaelis-Menten mechanism, similar to the oxidation of glucose on the immobilized GOD. From extrapolation of [S]- 1 = 0 in the plot of [M]-l vs. [S]-I, the maximum amount of galactose coordinated per unit surface area is calculated to be 2.3x 10 15 molecules cm- 2 (Table 1). Since one GAOD enzyme is known to coordinate one galactose molecule, it follows that there is an equivalent amount of the immobilized GAOD. As shown in Table 1, the number of Cu ions accommodated per unit surface area was 5.9 X 10 15
464
atoms cm - 2. Thus there were more Cu ions than active sites by a factor of 2.6. In other words, approximately 40% of the Cu ions work effectively to constitute the active sites, whereas the rest can be considered to be deactivated during immobilization by the silan coupling process. As shown in Fig. 2, complete recovery of the activity was not achieved in the Cu-apoenzyme (85% of the GAOD level). The lost 15% activity is apparently associated with the destruction of the conformation by attack of the chelating agent. A similar situation of partial recovery (87%) holds for the coordination of galactose (Fig. 3). The fact that the extent of recovery is nearly the same between the catalytic activity and the coordination ability indicates that almost all the coordinated galactose undergoes catalytic reactions, and thus the coordination sites turn out to be reaction sites as well. In conclusion, the SAW method is useful for analyzing the role of metal ions in immobilized metalloenzymes and the behavior of coordinated species during catalytic reactions.
Acknowledgement
This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science and Culture.
References 1 2 3 4 5 6 7 8
H. Wohltjen, Sensors and Actuators, 5 (1984) 307. J. Janata, Principles of Chemical Sensors, Plenum, New York and London, 1989, p. 69. Y. Inoue, Y. Kato and K. Sato, J. Chem. Soc., Faraday Trams. 1, 88 (1992) 449. S. J. Martin and A. J. Ricco, Sensors and Actuators, A21-A23 (1990) 712. J. A. Groetsch III and R. E. Dessy, J. Appl. Polym. Sci., 28 (1983) 161. Y. Inoue, M. Matsukawa and K. Sato, J. Am. Chem. Soc., 111 (1989) 8965. Y. Inoue and M. Matsukawa, J. Chem. Soc., Chern: Commun., (1990) 296. I. Satoh, T. Kasahara and M. Goi, Sensors and Actuators, B1 (1990) 499.
459
Application of an SH surface acoustic wave to the analysis of catalysis on immobilized galactose oxidase Yasunobu Inoue" and Yoshihiro Kato Department of Chemistry and Analysis Center, Nagaoka University of Technology, Nagaoka, Niigata 940-21 (Japan)
Abstract The surface acoustic wave (SAW) method, combined with measurements of reaction rates, was employed to investigate the role of the metal ion in the catalysis of a metalloenzyme. A device generating a shear horizontal leaky SAW was fabricated on which galactose oxidase (GAOD) was immobilized. The removal of Cu ions from immobilized GAOD to produce an inactive apoenzyme and its reverse process were monitored by changes in the SAW frequency. From the response in the SAW frequency during the oxidation of galactose, the formation of an enzyme-substrate complex was confirmed for GAOD, whereas the apoenzyme was found to have no capability of coordinating galactose, which was responsible for deactivation. Zn and Co ions were also accommodated in the apoenzyme to almost the same extent as Cu ions, but had no capability of coordinating galactose. It is concluded that the Cu ions control the coordination of galactose.
Introduction In an attempt to develop a method for analyzing the behavior of catalytic reactions on solid surfaces, we have focused on the great promise shown by surface acoustic waves (SAWs). Since the resonant frequency of the SAW is very sensitive to changes in mass loaded on the propagation path of the wave [1, 2 J, it is possible to measure even a trace amount of adsorbed species on the surface. Furthermore, a fact that measurements of the SAW frequency can be made independently of measurements of reaction rates permits determination of the amounts of the adsorbed species during the catalytic reactions. Recently we showed the availability of a shear horizontal (SH) leaky SAW to wave propagation in liquid phases and successfully applied it to examine the reaction mechanism of glucose oxidation on the immobilized glucose oxidase [3 J. Galactose oxidase is a metalloenzyme involving Cu ions as an essential metal ion and is active for the oxidation of galactose to produce galactohexodialdose and hydrogen peroxide. The removal of the Cu ions converts the active enzyme to an apoenzyme that has no catalytic activity. As for the role of the Cu ions, at least three questions remain unsolved. First, there *Author to whom correspondence should be addressed.
0304-5102/92/$5.00
© 1992 - Elsevier Sequoia. All rights reserved
460
are two possibilities for deactivation caused by Cu elimination, from a mechanistic point of view. One is that the apoenzyme itself has no capability of coordinating galactose, such that there is no chance for the reaction to proceed further. The other is that the apoenzyme is able to coordinate the substrate but loses the activity of oxidizing the coordinated species. The second question is whether or not metal ions other than Cu ions can be accommodated in the apoenzyme, and the third question is, if the accommodation takes place, whether or not the resulting enzymes have the ability to coordinate galactose. Finding answers to these questions will lead to understanding of the role of the Cu ions in the catalytic functions of GAOD. For this purpose, in addition to measurements of the catalytic activity, we need to monitor directly the amounts of the substrate coordinated on immobilized metalloenzymes and apoenzymes during the catalytic reactions, but so far there have been few studies employing such an approach [4, 5]. In the present study, the shear horizontal leaky SAW method was applied to the system of the immobilized GAOD and the apoenzyme.
Experimental The SAW device was the same as previously used [3]. The input and output interdigital transducer (IDT) electrodes, having a 200 JLm periodicity [6, 7], were fabricated photolithographically on a 36°-rotatedY-cut single crystal of LiTa03. The center frequency was 20.95 MHz in air. The catalyst was prepared by immobilizing galactose oxidase (GAOD) (Sigma Chemical) at pH = 7 in phosphate buffer solutions on the SAW device surface functionalized by a silane coupling agent, 3-aminopropyltriethoxysilane, and then by glutalaldehyde. The resonant SAW frequency of the catalyst was amplified by a feedback path in an oscillator loop and was monitored by a frequency counter. The SAW frequency change was converted to mass units according to the equation previously derived [3]. The catalyst was mounted on a special reaction cell for the simultaneous measurement of the SAW frequency and the reaction rates. The reaction rates at 298 K were determined from the amount of hydrogen peroxide produced, which was measured with a threeelectrode system using a potentiostat. The anode potential of the Pt working electrode was kept at +0.6 V vs. SCE.
Results After the resonant SAW frequency of the GAOD immobilized catalyst reached a constant level in the phosphate buffer solution at pH 8, a solution of a chelating agent, N ,N-diethyl-dithiocarbamic acid sodium salt, was added at a concentration of 10 roM. As shown in Fig. 1, the SAW frequency increased gradually and attained a saturation level. The total change in frequency amounted to 65 Hz. The solution examined was replaced by a fresh buffer
461 (a)
1
20 HZ
N
:r:
:;-
Chelating
agent
I 0
2
3
tlmin (I)
(b)
(3)
1.2 N
:r:
-
«c
Cu ion
OB Gal3.ctose
_ 0.4
1
20 Hz
( (2)
0
0
4 t /min
6
0
1 t/min
2
Fig. 1. Changes in SAW frequency: (a) addition of the chelating agent to a GAOD system; (b) after (a), addition of copper sulfate aqueous solution. Fig. 2. Catalytic activity for the oxidation of galactose on three enzymes: (1) GAOD; (2) apoenzyme; (3) Cu-apoenzyme. Conditions: pH 6, in 0.1 M phosphate buffer solution.
solution, and then the aqueous solution of copper sulfate was introduced. The frequency attenuated monotonically until it reached a constant level with a decrease of 65 Hz. The increase in the SAW frequency caused by the addition of the chelating agent was compensated for by an equal decrease caused by the addition of Cu ion solutions. This reversible change reflects the removal of Cu ions by the chelating agent from GAOD to form an apoenzyme and the accommodation of Cu ions to form GAOD. (In order to differentiate it from as-immobilized, original GAOD, this GAOD is hereafter referred to as Gu-apoenzyme.) The catalytic activity for the oxidation of galactose was compared for the three immobilized enzymes, GAOD, the apoenzyme and the Gu-apoenzyme. As shown in Fig. 2, GAOD has a considerable activity, whereas there is no catalytic activity for the apoenzyme. For the Gu-apoenzyme, the activity was equal to up to 85% of that of the original GAOD. Figure 3 shows changes in the SAW frequency due to the coordination of galactose during the catalytic oxidation. When the galactose substrate was added at a concentration of 1 mM, the SAW frequency of the immobilized GAOD showed a rapid decrease and reached a saturation level of 53 Hz in a short time. On the other hand, a negligibly small frequency change occurred for the apoenzyme. The frequency shift in the Gu-apoenzyme was 46 Hz, which was 87% of the shift in the original GAOD. The ability of apoenzyme to accommodate metal ions other than Cu ions was examined for Zn and Go ions, whose ionic radii are close to that of Cu ions. Figure 4 shows changes in the SAW frequency when the immobilized apoenzyme was exposed to the aqueous solutions of cobalt chloride and
462
N
! :I:
Co ion
10Hz
! Galactose
N~-(2)
; 12~---------m
Zn ion
1
20 HZ
o t
5 Imin
10
o
2
4
t I min
6
Fig. 3. Response of the SAWfrequency upon the addition of galactose: (1) GAOD; (2) apoenzyme; (3) Cu-apoenzyme. Experimental conditions are the same as in Fig. 2. Fig. 4. Changes in the SAW frequency when immobilized apoenzyme is exposed to the solutions of Co and Zn ions.
TABLE 1
Properties of the apoenzyme accommodating various metal ions Metal ions
Amount of metal ions accommodated (atoms cm- z)
Maximum amount of galactose coordinated (molecules cm- z)
Catalytic activity
Cu z+
5.9x 10 15 5.7x 1015 6.1 X 10 15
2.3 x 10 15 0 0
active inactive inactive
Znz + Co z+
zinc chloride. Negative frequency shifts were observed, indicative of the formation of Co- and Zn-apoenzymes, respectively. There was no significant difference in the total frequency shifts between Co (63 Hz) and Zn (65 Hz) ions, and it should be noted that the magnitudes of the frequency changes were similar to that for Cu ion. When galactose was introduced into the Zn-apoenzyme system, there was little change in the SAW frequency. A negligibly small response of the SAW frequency upon the addition of galactose was also observed for the Co-apoenzyme. Table 1 summarizes the properties of the apoenzymes accommodating the various metal ions. Figure 5 shows the amounts of galactose adsorbed, [M], on the immobilized GAOD during the catalytic reaction as a function of the galactose concentration, [S]. At low concentrations, the value of [M] increased remarkably, followed by a less rapid increase, and attained an almost saturation level above 2 roM. A straight line was obtained when [M]-I was plotted against [S ]-1.
463
2
o
2
4
6
[5], Galactose I mM Fig. 5. Amount of galactose coordinated on immobilized GAOD as a function of galactose concentration. Conditions: pH 6, 0.1 M phosphate buffer solution.
Discussion The oxidation of galactose on immobilized GAOD has been examined mostly by measuring the amount of H 20 2 produced using electrochemical methods [8]; the behavior of the coordinated species was not well understood. The present SAW results clearly indicate that no coordination of galactose on the immobilized GAOD occurs in the absence of Cu metal ions. Thus it is logical to assume that lack of apoenzyme catalytic activity can be associated with loss of the capability to form an enzyme-substrate complex. As shown in Table 1, Cu, Zn and Co ions are all accommodated in the apoenzyme to a similar extent. This indicates that the metal-ion coordination sites of the apoenzyme have no specificity in the selection of these metal ions. On the other hand, only the Cu-apoenzyme showed activity for galactose coordination, in contrast to the absence of activity of the Zn- and Co-apoenzymes. From these results, it is concluded that the recognition of the substrate through coordination is essential for the functions of the metalloenzyme. Cu ions have a high potential to arrange the highly ordered structural conformation suitable for the coordination of galactose. In the previous study [3] using the SAW method for the oxidation of glucose on the immobilized glucose oxidase (GOD), the Michaelis constant was found to be in good agreement with the equilibrium constant of glucose coordination during the reaction, and thus it was shown that almost all the coordinated glucose molecules participated in the catalytic reaction and that the coordination sites homogeneously contributed to the active sites. The linear relationship between [M]- 1 and [S]- 1 observed here suggests that the oxidation of galactose on the immobilized GAOD proceeds according to the Michaelis-Menten mechanism, similar to the oxidation of glucose on the immobilized GOD. From extrapolation of [S]- 1 = 0 in the plot of [M]-l vs. [S]-I, the maximum amount of galactose coordinated per unit surface area is calculated to be 2.3x 10 15 molecules cm- 2 (Table 1). Since one GAOD enzyme is known to coordinate one galactose molecule, it follows that there is an equivalent amount of the immobilized GAOD. As shown in Table 1, the number of Cu ions accommodated per unit surface area was 5.9 X 10 15
464
atoms cm - 2. Thus there were more Cu ions than active sites by a factor of 2.6. In other words, approximately 40% of the Cu ions work effectively to constitute the active sites, whereas the rest can be considered to be deactivated during immobilization by the silan coupling process. As shown in Fig. 2, complete recovery of the activity was not achieved in the Cu-apoenzyme (85% of the GAOD level). The lost 15% activity is apparently associated with the destruction of the conformation by attack of the chelating agent. A similar situation of partial recovery (87%) holds for the coordination of galactose (Fig. 3). The fact that the extent of recovery is nearly the same between the catalytic activity and the coordination ability indicates that almost all the coordinated galactose undergoes catalytic reactions, and thus the coordination sites turn out to be reaction sites as well. In conclusion, the SAW method is useful for analyzing the role of metal ions in immobilized metalloenzymes and the behavior of coordinated species during catalytic reactions.
Acknowledgement
This work was supported by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science and Culture.
References 1 2 3 4 5 6 7 8
H. Wohltjen, Sensors and Actuators, 5 (1984) 307. J. Janata, Principles of Chemical Sensors, Plenum, New York and London, 1989, p. 69. Y. Inoue, Y. Kato and K. Sato, J. Chem. Soc., Faraday Trams. 1, 88 (1992) 449. S. J. Martin and A. J. Ricco, Sensors and Actuators, A21-A23 (1990) 712. J. A. Groetsch III and R. E. Dessy, J. Appl. Polym. Sci., 28 (1983) 161. Y. Inoue, M. Matsukawa and K. Sato, J. Am. Chem. Soc., 111 (1989) 8965. Y. Inoue and M. Matsukawa, J. Chem. Soc., Chern: Commun., (1990) 296. I. Satoh, T. Kasahara and M. Goi, Sensors and Actuators, B1 (1990) 499.