- Email: [email protected]
Membrane potential of Bombyx mori silk fibroin membrane induced by an immobilized enzyme reaction
167
Bioelectrochemistry and Bioenergetics, 26 (1991) 167-175 A section of J. Electroanal. Chem., and constituting Vol. 321 (1991) Elsevier Sequoia S.A., Lausanne
JEC BB 01383
Membrane potential of Bombyx mori silk fibroin membrane induced by an immobilized enzyme reaction Makoto Demura, Takashi Komura and Tetsuo Asakura * Faculty of Technology, Tokyo 184 (Japan) (Received
25 March
Tokyo University
of Agriculture
and Technology, Nakamachi
2-Chome,
Koganei,
1991)
Abstract The membrane potential of Bombyx mori silk fibroin membrane induced by the reaction of glucose oxidase (GO) which was immobilized in the silk fibroin membrane, has been measured. The membrane potential increased rapidly after addition of the substrate, glucose, and reached a plateau. The degree of response depended on the concentration of glucose. From direct observation of the 13C NMR spectra of the product of the enzyme reaction in the silk fibroin membrane, it was concluded that the gluconolactone generated from the glucose was completely converted to gluconic acid in the silk fibroin membrane. In addition, a greater pH difference between the membrane surfaces after the enzyme reaction was determined. It was found that the effective anionic fixed charge density of silk fibroin membrane decreased with increasing ghrconic acid concentration in the membrane.
INTRODUCTION
Silk fibroin prepared from Bombyx mori cocoons has been applied successfully as support for enzyme immobilization [l-12]. One of the merits of silk fibroin as an enzyme immobilization support is that the enzymes can be mildly entrapped into the silk fibroin membrane without any use of chemical reagents. Namely, the structural transition of the fibroin from random coil to anti-parallel P-sheet occurs readily upon physical treatment only, such as stretching, compressing, standing under high humidity and immersing in an alcohol aqueous solution, resulting in the simultaneous insolubilization of the membrane and the immobilization of enzyme [13,14]. In addition, a sensor assembled from an oxygen electrode using glucose oxidase (GO) immobilized within a silk fibroin membrane has been developed [9,111. a
l
To whom correspondence
0302-4598/91/$03.50
should be addressed. 0 1991 - Elsevier Sequoia
S.A. AU rights reserved
168
The membrane potential induced by the reaction in and/or on a membrane has been measured with some enzyme immobilized membranes [15-181. However, these need large amounts of enzyme and the structural effect of the enzyme supports on the membrane potential induced by the immobilized enzyme reaction was not discussed. In this paper, we will ,report the membrane potential of B. mori silk fibroin membrane induced by GO immobilized in the membrane. The degree of dissociation of the ionic product of the GO reaction in the membrane phase is determined by 13C nuclear magnetic resonance (NMR) spectroscopy. The basic mechanism of the generation of the membrane potential after the enzyme reaction will be deduced from the behavior of the products, that is, their influence on both the enzyme reaction and the structure ‘of the silk fibroin membrane as a support. The generation of the mernbrane potential of the silk fibroin membrane as regards the concentration potential with alkaline ions has been investigated previously [19,20]. It is found that generation of the membrane potential by the reaction of GO immobilized in the silk fibroin membrane is due to the production of ionic species by the immobilized enzyme. EXPERIMEhTAL
Preparation of the GO immobilized silk fibroin membrane
The silk fibroin aqueous solution was prepared from Bombyx mori cocoons as described previously [9,21]. 3 w/v% of the silk fibroin solution was mixed mildly with a given amount of GG (derived from Aspergillus niger, WAKO Pure Reagents Co.) aqueous solution. This~fr&tnre waseast on an acrylic plastic plate, and dried at 20°C under 50% relative humidity for 48 h. The dried membrane was insolubilized by immersion in 80%. methanol aqueous solution for 1 h, and then washed with distilled water’[ll]. ?he GO irhrnobilized silk fibrom membrane was stored in a dry state at 4 o C until measurements. The thickness of the membrane was ca. 50 pm. Me&urement
of metirahe
potential
The GO immobilized silk fibroh membrane was sufficiently swollen in distilled water and then the membrane was set positioned between the cells with silicone sheets as shown in Fig. 1. The voiume of each compartment was 80 ml and the effective reaction area of the membrane was 3.14 cm2. An electrometer (Model TR 8652, Advantest Co., Japan) was connected to each compartment with Ag/AgCl electrodes through salt bridges. After the cell was filled with 0.1 mM potassium phosphate buffer,,solution (RH 7), a: given amount of glucose aqueous solution (1 M) was added to, the ieft. com&rtment and the potential difference between the two compartments was measured at 25°C. ‘% NMR rrieastir&ment 13C NMRspect’fa were recorded at 25°C tith
a JEOL FX-90Q NMR spectrometer operating at 22.49 MHz. ‘f’he product of the Go reaction, gluconolactone, was
169
Fig. 1. Scheme of the apparatus for measurement of the membrane potential with the GO reaction in the silk fibroin membrane. (1) Right compartment, (2) left compartment, (3) salt bridge, (4) substrati: solution, (5) GO immobilized silk fibroin membrane, (6) Stirrer.
measured in the aqueous solution, where the pH was adjusted with 0.1 M sodium hydroxide and hydrochloric acid aqueous solutions. Moreover, the 13C NMR spectrum of the product in the silk fibroin membrane after the GO reaction was observed directly. After the GO immobilized membrane was immersed in 0.5 ti glucose aqueous solution (pH 7) for 30 min, the membrane was picked up, and then measured.
Measurement of pH after the GO reaction The pH change of the GO immobilized silk fibroin membrane after the enzyme reaction was measured by the following method. The GO immobilized silk fibroin membrane was dipped in 0.1 mM potassium phosphate buffer (pH 7) overnight. Then the membrane was attached to a glass pH electrode (Model TP-101, Toko Chemical Laboratories Co., Tokyo, Japan) with a nylon net and an O-ring. A given amount of glucose solution (1 M) was injected into the reaction chamber with the assembled pH electrode. Then the pH change was recorded. The change of pH in the silk fibroin membrane was also measured with a pH indicator, 3-sulfo-2,6-dichloro-3’,3”-dimethyl-4’-hydroxyfuchsone-5’,5”-dicarboxylic acid, trisodium salt (Chromazurol S, Dojindo Laboratories, Kumamoto, Japan), by monitoring the absorbance at 430 nm by means of an UV spectrometer (U-3200, Hitachi) equipped with a thermostated cell holder. Determination of the fixed charge density of the silk fibroin membrane The membrane potential was measured at a given pH by changing tion of KC1 in each compartment while maintaining the concentration I
the conceni:l;aratio of KC1
170
% C-J dL
./“ 3mi n
AE
----
----______
I - _____
GLUCOSE
Fig. 2. Typical response of the membrane potential with the reaction of 2% GO immobilized fibroin membrane after injection of glucose (final concentration = 1 mM).
at 2 : 1. The fixed charge density potential as a function of pH [19]. RESULTS
was calculated
using
the observed
in the silk
membrane
AND DISCUSSION
Membrane potential induced by the GO reaction The membrane potential of the GO immobilized silk fibroin membrane during the enzyme reaction was measured. Figure 2 depicts a typical response of the membrane potential of the GO immobilized silk fibroin membrane after injection of the substrate. In this case, the concentration of potassium phosphate in each cell was the same and did not change during the injection. It has been reported previously that no leakage of GO from the silk fibroin membrane was observed [5]. The membrane potential increased gradually and reached a plateau, as shown in Fig. 2. In the absence of GO, no membrane potential was observed after the injection of glucose solution. Figure 3 shows a plot of AE, the membrane potential in the steady state, against the glucose concentration of the solution in the left compartment. The potential increased with increasing glucose concentration in the range of 0 to 5 mM. Previously, the membrane potential of an enzyme immobilized membrane had been measured with an albumin membrane bearing urease [22] and
$ 1 !
4t
o-
3-
0’
0 0
0’
%O/ I-
Fig. 3. Calibration fibroin membrane.
curve of glucose concentration
with the reaction of 2% GO immobilized
in the silk
171 CHzOH
Gluconolactone
200
150
100 ppmfmmext.lMs
50
Fig. 4. “C NMR spectra of products of the GO reaction. Gluconolactone aqueous solution at two pHs: (A) 6.8 and (ES) 3.0. (C) Porous silk fibroin membrane after immobilized GO reaction. Cl-C6: Gluconic acid, (A) gluconolactone.
with acetylcholinesterase immobilized ossein gelatin membrane or albumin membrane [181. The former was measured with different phosphate buffer concentrations on each side of the membrane. The membrane potential changes were based on the reciprocal effect between enzyme activity and the appearance of ions after the enzyme reaction, i.e., a modification of the local pH and a feedback action of buffer diffusion from the outside solution. The latter study also pointed out the effect on the membrane potential of ionic species produced by the enzyme. However, it is insufficient to understand the detailed information concerning the effect of products on the enzyme support. ‘% NMR observation of the GO reaction in the silk fibroin membrane It is necessary to pay attention to the ionic product, gluconic acid, because the ionic product in the membrane may generate the ion diffusion in the membrane and/or the local pH change in the membrane. In addition, the fine structure of the silk fibroin membrane is considered to be affected by the ionic product. However, the state of the ionic species produced in the membrane was not observed directly in spite of the importance of the ionic product. Therefore, we observed the state of the products after the enzyme reaction in the silk fibroin membrane using 13C NMR. Figures 4A and B show 13C NMR spectra of gluconolactone aqueous solution at two pHs, 6.8 and 3.0, respectively. All peak intensities attributable to gluconic acid are
172
16 t
> E
n-
Q-
\
cr .: i 8-
/
n’
A’
0
9)
5 L 4-
n
? Y
/
6
d 8
OI
1
I
1,25 CP
/
Q---
I
I
1.5
1,75
I
2,o
Cl
Fig. 5. Concentration vs. membrane potential of the silk fibroin membrane with gluconic acid produced from gluconolactone, and of KCI. The concentration ratio (c*/c,) was changed from 1 to 2. (0) Gluconic acid (c, = 0.01 M), ( 0) gluconic acid (c, = 0.001 M), (A) KC1 (c, = 0.01 M).
stronger than those of gluconolactone carbons even at pH = 3.0. Further, Fig. 4C depicts the ‘3C NMR spectrum of the product in porous GO immobilized membrane generated by the immobilized enzyme in the membrane. The peaks can be assigned exclusively to the carbons of gluconic acid, although the peaks are broad because of the low mobility of the products rather than the high mobility of those in solution. Under these conditions, the carbons of silk fibroin were not observed. Thus, it is concluded that gluconolactone, which is the product of the GO reaction, is rapidly hydrolyzed to gluconic acid in the silk fibroin membrane. In addition, pH titration of gluconic acid suggested that the pH in the GO immobilized membrane is shifted to the acidic side by the enzyme reaction.
Effects of gluconic acid on the membrane potential of the silk fibroin membrane Figure 5 depicts the concentration membrane potential of the silk fibroin membrane with gluconic acid produced from gluconolactone. This indicates that the addition of gluconic acid to one side of the silk fibroin membrane generates a membrane potential and that the polarity is the same as with KCl. However, the membrane potential differences using gluconic acid are small compared with those using KCl, suggesting an effect of pH change on the membrane potential. When the gluconic acid is preferentially produced with the immobilized GO reaction at one side of the membrane, this ionic species may diffuse across the silk fibroin membrane. It is expected that a pH change occurs locally, caused by the presence of gluconic acid. Such a pH change on the membrane surface and in the
173
Fig. 6. Plots of the rate of pH change of the GO immobilized silk fibroin membrane against the concentration of glucose. (0) 0.2% GO, (A) 1.0%GO, (0) 2.0% Go.
silk fibroin membrane was measured with both a pH glass electrode and UV methods. The former method gives pH information concerning the surface not in contact with the substrate directly, while the latter one gives the information of pH change on the other membrane surface and in the membrane. Figure 6 shows the relationship between the rate of pH change in the steady state and the glucose concentration in the reaction chamber. During the GO reaction, the pH on the membrane surface not in contact with the substrate was shifted to the acidic side and the rate of the pH change depended on the concentration of glucose in the reaction chamber. The plots are regarded as straight lines, the slopes of which increase with increasing content of immobilized GO. The slopes are 0.33 x 10e3, 4.17 X 1O-3 and 12.3 X low3 ApH min-’ mM_‘, for GCI contents of 0.28, 1.0% and 2.0% respectively, which correspond to the amount of gluconic acid produced by the GO reaction in the silk fibroin membrane. The pH change on the membrane surface in contact with the substrate was also estimated, from the UV method. The absorbance of a pH indicator, Chromazurol S, in the presence of both a GO immobilized membrane and glucose was recorded continuously and then the initial rate of the pH change with the enzyme reaction was determined by using the calibration curve. For a GO content of 0.2%, the rate was 9.8 X lOA ApH min-’ mM- I. Thus, the pH change at the surface in contact with the substance was much larger than that observed using the pH electrode. This result indicates that the production of gluconic acid by the immobilized GO reaction may change the pH of both the inner and surface regions of the membrane and that the concentration of product at both surfaces of the membrane is not equal. These results agree with the concentration membrane potential with gluconic acid, the 13C NMR observation of gluconic acid and also with a theoretical study of the general transport of the products of an enzyme membrane [23]. The non-uniform distribution of the ionic species over the two membrane surfaces is considered to result not only from ion diffusion but also from the asymmetric change of the microenvironment of the silk fibroin membrane (see below).
174
Fig. 7. Fixed charge density of various silk fibroin membranes as a function of pH. Mcllvaine buffer concentration was 0.01 M. (0) GO immobilized silk fibroin membrane, (0) silk fibroin membrane.
The effective fixed charge density of the silk fibroin is expected to be affected by the gluconic acid during the enzyme reaction in the membrane. Figure 7 shows plots of the fixed charge density as a function of pH. The silk fibroin membrane has net positive charges in the acidic region and negative ones in the basic region relative to the isoelectric point, which is pH 4.3 for the original silk fibroin membrane and pH 4.5 for the GO immobilized silk fibroin membrane. In particular, the effective fixed charge density changed remarkably in the pH range from 7 to the pH of the isoelectric point. Therefore, this change relates to the generation of the Donnan potential on each surface of the GO immobilized silk fibroin membrane. In addition, the fine structure of the silk fibroin membrane is expected to be influenced by the interaction between gluconic acid and the silk fibroin enzyme support, suggesting an effect of membrane structure on the membrane potential coupled to the immobilized enzyme reaction. In general, the membrane potential difference with an artificial fixed charge membrane such as a silk fibroin membrane is expressed as follows [24]: AE = AE Donnan+ A Ediffusion, where A EDonnan and A Ediffusion are the Donnan and diffusion potentials, respectively. The former potential is the sum of the two surface potentials at both sides of the membrane and the latter is the diffusion potential in the membrane. The enzyme reaction in the membrane may cause a change in both AE Donnan and A Eciiflusion. It is particularly important to reveal the behavior of ionic species after the enzyme reaction in the silk fibroin membrane. It will therefore be necessary to investigate the mobility of ionic species in the silk fibroin membrane and the interaction between the product and the silk fibroin in order to improve the response and to design an effective structure for the silk fibroin membrane as an enzyme support.
175 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
L. Gras&, D. Cordier and A. Ville, Biotechnol. Bioeng., 19 (1977) 611. L. Grasset, D. Cordier and A. Ville, Process B&hem., 14 (1979) 2. S. Miyairi, M. Sugiura and F. Fukui, Agric. Biol. Chem., 42 (1978) 1661. D. Cordier, R. Couturier, L. Grasset and A. Ville, Enzyme Microb. Technol., 4 (1982) 249. A. Kuzuhara, T. Asakura, R. Tomoda and T. Matsunaga, J.Biotechnd., 5 (1987) 199. T. Asakura, Bioindustry, 4 (1987) 36. T. Asakura, H. Yoshimizu, A. Kuzuhara and T. Matsunaga, J. Seric. Sci. Jpn., 57 (1988) 203. T. Asakura, J. Kanetake and M. Demura, J. Polym. Plast. Technol. Eng., 28 (1989) 453. M. Demura and T. Asakura, Biotechnol. Bioeng., 33 (1989) 598. M. Demura, T. Asakura, E. Nakamura and H. Tamura, J. Biotechnol., 10 (1989) 113. M. Demura, T. Asakura and T. Kuroo, Biosensors, 4 (1989) 361. H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 127. H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 1745. T. Asakura, H. Yoshimizu and M. Kakizaki, Biotechnol. Bioeng., 35 (1990) 511. R. Tor and A. Freeman, Anal. Chem., 58 (1979) 1042. T. Shinbo, M. Sugiura and N. Kamo, Anal. Chem., 51 (1979) 100. R.H. William and H.B. HalsaIl, Anal. Chem., 57 (1985) 1321A. A. Friboulet and D. Thomas, Biophys. Chem., 16 (1982) 153. M. Sugiura, Nippon Nogei Kagaku Kaishi, 47 (1973) 563. M. Demura, A. Kitamura, A. Shibamoto and T. Asakura, J. Appl. Polym. Sci., 36 (1988) 535. T. Asakura, Y. Watanabe, A. Uchida and H. Minagawa, Macromolecules, 17 (1984) 1075. A. David, M. Metayer, D. Thomas and G. Broum, J. Membrane Biol., 18 (1974) 113. T. Ciftci and W.R. Vieth, J. Mol. Catal., 8 (1980) 455. Y. Kobatake, K. Kurihara N. Kamo in T. Takamura and A. Kozawa (Eds.), Surface Electrochemistry. Advances and Concepts, Japan Scientific Societies Press, Tokyo, 1978, p. 1.
Bioelectrochemistry and Bioenergetics, 26 (1991) 167-175 A section of J. Electroanal. Chem., and constituting Vol. 321 (1991) Elsevier Sequoia S.A., Lausanne
JEC BB 01383
Membrane potential of Bombyx mori silk fibroin membrane induced by an immobilized enzyme reaction Makoto Demura, Takashi Komura and Tetsuo Asakura * Faculty of Technology, Tokyo 184 (Japan) (Received
25 March
Tokyo University
of Agriculture
and Technology, Nakamachi
2-Chome,
Koganei,
1991)
Abstract The membrane potential of Bombyx mori silk fibroin membrane induced by the reaction of glucose oxidase (GO) which was immobilized in the silk fibroin membrane, has been measured. The membrane potential increased rapidly after addition of the substrate, glucose, and reached a plateau. The degree of response depended on the concentration of glucose. From direct observation of the 13C NMR spectra of the product of the enzyme reaction in the silk fibroin membrane, it was concluded that the gluconolactone generated from the glucose was completely converted to gluconic acid in the silk fibroin membrane. In addition, a greater pH difference between the membrane surfaces after the enzyme reaction was determined. It was found that the effective anionic fixed charge density of silk fibroin membrane decreased with increasing ghrconic acid concentration in the membrane.
INTRODUCTION
Silk fibroin prepared from Bombyx mori cocoons has been applied successfully as support for enzyme immobilization [l-12]. One of the merits of silk fibroin as an enzyme immobilization support is that the enzymes can be mildly entrapped into the silk fibroin membrane without any use of chemical reagents. Namely, the structural transition of the fibroin from random coil to anti-parallel P-sheet occurs readily upon physical treatment only, such as stretching, compressing, standing under high humidity and immersing in an alcohol aqueous solution, resulting in the simultaneous insolubilization of the membrane and the immobilization of enzyme [13,14]. In addition, a sensor assembled from an oxygen electrode using glucose oxidase (GO) immobilized within a silk fibroin membrane has been developed [9,111. a
l
To whom correspondence
0302-4598/91/$03.50
should be addressed. 0 1991 - Elsevier Sequoia
S.A. AU rights reserved
168
The membrane potential induced by the reaction in and/or on a membrane has been measured with some enzyme immobilized membranes [15-181. However, these need large amounts of enzyme and the structural effect of the enzyme supports on the membrane potential induced by the immobilized enzyme reaction was not discussed. In this paper, we will ,report the membrane potential of B. mori silk fibroin membrane induced by GO immobilized in the membrane. The degree of dissociation of the ionic product of the GO reaction in the membrane phase is determined by 13C nuclear magnetic resonance (NMR) spectroscopy. The basic mechanism of the generation of the membrane potential after the enzyme reaction will be deduced from the behavior of the products, that is, their influence on both the enzyme reaction and the structure ‘of the silk fibroin membrane as a support. The generation of the mernbrane potential of the silk fibroin membrane as regards the concentration potential with alkaline ions has been investigated previously [19,20]. It is found that generation of the membrane potential by the reaction of GO immobilized in the silk fibroin membrane is due to the production of ionic species by the immobilized enzyme. EXPERIMEhTAL
Preparation of the GO immobilized silk fibroin membrane
The silk fibroin aqueous solution was prepared from Bombyx mori cocoons as described previously [9,21]. 3 w/v% of the silk fibroin solution was mixed mildly with a given amount of GG (derived from Aspergillus niger, WAKO Pure Reagents Co.) aqueous solution. This~fr&tnre waseast on an acrylic plastic plate, and dried at 20°C under 50% relative humidity for 48 h. The dried membrane was insolubilized by immersion in 80%. methanol aqueous solution for 1 h, and then washed with distilled water’[ll]. ?he GO irhrnobilized silk fibrom membrane was stored in a dry state at 4 o C until measurements. The thickness of the membrane was ca. 50 pm. Me&urement
of metirahe
potential
The GO immobilized silk fibroh membrane was sufficiently swollen in distilled water and then the membrane was set positioned between the cells with silicone sheets as shown in Fig. 1. The voiume of each compartment was 80 ml and the effective reaction area of the membrane was 3.14 cm2. An electrometer (Model TR 8652, Advantest Co., Japan) was connected to each compartment with Ag/AgCl electrodes through salt bridges. After the cell was filled with 0.1 mM potassium phosphate buffer,,solution (RH 7), a: given amount of glucose aqueous solution (1 M) was added to, the ieft. com&rtment and the potential difference between the two compartments was measured at 25°C. ‘% NMR rrieastir&ment 13C NMRspect’fa were recorded at 25°C tith
a JEOL FX-90Q NMR spectrometer operating at 22.49 MHz. ‘f’he product of the Go reaction, gluconolactone, was
169
Fig. 1. Scheme of the apparatus for measurement of the membrane potential with the GO reaction in the silk fibroin membrane. (1) Right compartment, (2) left compartment, (3) salt bridge, (4) substrati: solution, (5) GO immobilized silk fibroin membrane, (6) Stirrer.
measured in the aqueous solution, where the pH was adjusted with 0.1 M sodium hydroxide and hydrochloric acid aqueous solutions. Moreover, the 13C NMR spectrum of the product in the silk fibroin membrane after the GO reaction was observed directly. After the GO immobilized membrane was immersed in 0.5 ti glucose aqueous solution (pH 7) for 30 min, the membrane was picked up, and then measured.
Measurement of pH after the GO reaction The pH change of the GO immobilized silk fibroin membrane after the enzyme reaction was measured by the following method. The GO immobilized silk fibroin membrane was dipped in 0.1 mM potassium phosphate buffer (pH 7) overnight. Then the membrane was attached to a glass pH electrode (Model TP-101, Toko Chemical Laboratories Co., Tokyo, Japan) with a nylon net and an O-ring. A given amount of glucose solution (1 M) was injected into the reaction chamber with the assembled pH electrode. Then the pH change was recorded. The change of pH in the silk fibroin membrane was also measured with a pH indicator, 3-sulfo-2,6-dichloro-3’,3”-dimethyl-4’-hydroxyfuchsone-5’,5”-dicarboxylic acid, trisodium salt (Chromazurol S, Dojindo Laboratories, Kumamoto, Japan), by monitoring the absorbance at 430 nm by means of an UV spectrometer (U-3200, Hitachi) equipped with a thermostated cell holder. Determination of the fixed charge density of the silk fibroin membrane The membrane potential was measured at a given pH by changing tion of KC1 in each compartment while maintaining the concentration I
the conceni:l;aratio of KC1
170
% C-J dL
./“ 3mi n
AE
----
----______
I - _____
GLUCOSE
Fig. 2. Typical response of the membrane potential with the reaction of 2% GO immobilized fibroin membrane after injection of glucose (final concentration = 1 mM).
at 2 : 1. The fixed charge density potential as a function of pH [19]. RESULTS
was calculated
using
the observed
in the silk
membrane
AND DISCUSSION
Membrane potential induced by the GO reaction The membrane potential of the GO immobilized silk fibroin membrane during the enzyme reaction was measured. Figure 2 depicts a typical response of the membrane potential of the GO immobilized silk fibroin membrane after injection of the substrate. In this case, the concentration of potassium phosphate in each cell was the same and did not change during the injection. It has been reported previously that no leakage of GO from the silk fibroin membrane was observed [5]. The membrane potential increased gradually and reached a plateau, as shown in Fig. 2. In the absence of GO, no membrane potential was observed after the injection of glucose solution. Figure 3 shows a plot of AE, the membrane potential in the steady state, against the glucose concentration of the solution in the left compartment. The potential increased with increasing glucose concentration in the range of 0 to 5 mM. Previously, the membrane potential of an enzyme immobilized membrane had been measured with an albumin membrane bearing urease [22] and
$ 1 !
4t
o-
3-
0’
0 0
0’
%O/ I-
Fig. 3. Calibration fibroin membrane.
curve of glucose concentration
with the reaction of 2% GO immobilized
in the silk
171 CHzOH
Gluconolactone
200
150
100 ppmfmmext.lMs
50
Fig. 4. “C NMR spectra of products of the GO reaction. Gluconolactone aqueous solution at two pHs: (A) 6.8 and (ES) 3.0. (C) Porous silk fibroin membrane after immobilized GO reaction. Cl-C6: Gluconic acid, (A) gluconolactone.
with acetylcholinesterase immobilized ossein gelatin membrane or albumin membrane [181. The former was measured with different phosphate buffer concentrations on each side of the membrane. The membrane potential changes were based on the reciprocal effect between enzyme activity and the appearance of ions after the enzyme reaction, i.e., a modification of the local pH and a feedback action of buffer diffusion from the outside solution. The latter study also pointed out the effect on the membrane potential of ionic species produced by the enzyme. However, it is insufficient to understand the detailed information concerning the effect of products on the enzyme support. ‘% NMR observation of the GO reaction in the silk fibroin membrane It is necessary to pay attention to the ionic product, gluconic acid, because the ionic product in the membrane may generate the ion diffusion in the membrane and/or the local pH change in the membrane. In addition, the fine structure of the silk fibroin membrane is considered to be affected by the ionic product. However, the state of the ionic species produced in the membrane was not observed directly in spite of the importance of the ionic product. Therefore, we observed the state of the products after the enzyme reaction in the silk fibroin membrane using 13C NMR. Figures 4A and B show 13C NMR spectra of gluconolactone aqueous solution at two pHs, 6.8 and 3.0, respectively. All peak intensities attributable to gluconic acid are
172
16 t
> E
n-
Q-
\
cr .: i 8-
/
n’
A’
0
9)
5 L 4-
n
? Y
/
6
d 8
OI
1
I
1,25 CP
/
Q---
I
I
1.5
1,75
I
2,o
Cl
Fig. 5. Concentration vs. membrane potential of the silk fibroin membrane with gluconic acid produced from gluconolactone, and of KCI. The concentration ratio (c*/c,) was changed from 1 to 2. (0) Gluconic acid (c, = 0.01 M), ( 0) gluconic acid (c, = 0.001 M), (A) KC1 (c, = 0.01 M).
stronger than those of gluconolactone carbons even at pH = 3.0. Further, Fig. 4C depicts the ‘3C NMR spectrum of the product in porous GO immobilized membrane generated by the immobilized enzyme in the membrane. The peaks can be assigned exclusively to the carbons of gluconic acid, although the peaks are broad because of the low mobility of the products rather than the high mobility of those in solution. Under these conditions, the carbons of silk fibroin were not observed. Thus, it is concluded that gluconolactone, which is the product of the GO reaction, is rapidly hydrolyzed to gluconic acid in the silk fibroin membrane. In addition, pH titration of gluconic acid suggested that the pH in the GO immobilized membrane is shifted to the acidic side by the enzyme reaction.
Effects of gluconic acid on the membrane potential of the silk fibroin membrane Figure 5 depicts the concentration membrane potential of the silk fibroin membrane with gluconic acid produced from gluconolactone. This indicates that the addition of gluconic acid to one side of the silk fibroin membrane generates a membrane potential and that the polarity is the same as with KCl. However, the membrane potential differences using gluconic acid are small compared with those using KCl, suggesting an effect of pH change on the membrane potential. When the gluconic acid is preferentially produced with the immobilized GO reaction at one side of the membrane, this ionic species may diffuse across the silk fibroin membrane. It is expected that a pH change occurs locally, caused by the presence of gluconic acid. Such a pH change on the membrane surface and in the
173
Fig. 6. Plots of the rate of pH change of the GO immobilized silk fibroin membrane against the concentration of glucose. (0) 0.2% GO, (A) 1.0%GO, (0) 2.0% Go.
silk fibroin membrane was measured with both a pH glass electrode and UV methods. The former method gives pH information concerning the surface not in contact with the substrate directly, while the latter one gives the information of pH change on the other membrane surface and in the membrane. Figure 6 shows the relationship between the rate of pH change in the steady state and the glucose concentration in the reaction chamber. During the GO reaction, the pH on the membrane surface not in contact with the substrate was shifted to the acidic side and the rate of the pH change depended on the concentration of glucose in the reaction chamber. The plots are regarded as straight lines, the slopes of which increase with increasing content of immobilized GO. The slopes are 0.33 x 10e3, 4.17 X 1O-3 and 12.3 X low3 ApH min-’ mM_‘, for GCI contents of 0.28, 1.0% and 2.0% respectively, which correspond to the amount of gluconic acid produced by the GO reaction in the silk fibroin membrane. The pH change on the membrane surface in contact with the substrate was also estimated, from the UV method. The absorbance of a pH indicator, Chromazurol S, in the presence of both a GO immobilized membrane and glucose was recorded continuously and then the initial rate of the pH change with the enzyme reaction was determined by using the calibration curve. For a GO content of 0.2%, the rate was 9.8 X lOA ApH min-’ mM- I. Thus, the pH change at the surface in contact with the substance was much larger than that observed using the pH electrode. This result indicates that the production of gluconic acid by the immobilized GO reaction may change the pH of both the inner and surface regions of the membrane and that the concentration of product at both surfaces of the membrane is not equal. These results agree with the concentration membrane potential with gluconic acid, the 13C NMR observation of gluconic acid and also with a theoretical study of the general transport of the products of an enzyme membrane [23]. The non-uniform distribution of the ionic species over the two membrane surfaces is considered to result not only from ion diffusion but also from the asymmetric change of the microenvironment of the silk fibroin membrane (see below).
174
Fig. 7. Fixed charge density of various silk fibroin membranes as a function of pH. Mcllvaine buffer concentration was 0.01 M. (0) GO immobilized silk fibroin membrane, (0) silk fibroin membrane.
The effective fixed charge density of the silk fibroin is expected to be affected by the gluconic acid during the enzyme reaction in the membrane. Figure 7 shows plots of the fixed charge density as a function of pH. The silk fibroin membrane has net positive charges in the acidic region and negative ones in the basic region relative to the isoelectric point, which is pH 4.3 for the original silk fibroin membrane and pH 4.5 for the GO immobilized silk fibroin membrane. In particular, the effective fixed charge density changed remarkably in the pH range from 7 to the pH of the isoelectric point. Therefore, this change relates to the generation of the Donnan potential on each surface of the GO immobilized silk fibroin membrane. In addition, the fine structure of the silk fibroin membrane is expected to be influenced by the interaction between gluconic acid and the silk fibroin enzyme support, suggesting an effect of membrane structure on the membrane potential coupled to the immobilized enzyme reaction. In general, the membrane potential difference with an artificial fixed charge membrane such as a silk fibroin membrane is expressed as follows [24]: AE = AE Donnan+ A Ediffusion, where A EDonnan and A Ediffusion are the Donnan and diffusion potentials, respectively. The former potential is the sum of the two surface potentials at both sides of the membrane and the latter is the diffusion potential in the membrane. The enzyme reaction in the membrane may cause a change in both AE Donnan and A Eciiflusion. It is particularly important to reveal the behavior of ionic species after the enzyme reaction in the silk fibroin membrane. It will therefore be necessary to investigate the mobility of ionic species in the silk fibroin membrane and the interaction between the product and the silk fibroin in order to improve the response and to design an effective structure for the silk fibroin membrane as an enzyme support.
175 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
L. Gras&, D. Cordier and A. Ville, Biotechnol. Bioeng., 19 (1977) 611. L. Grasset, D. Cordier and A. Ville, Process B&hem., 14 (1979) 2. S. Miyairi, M. Sugiura and F. Fukui, Agric. Biol. Chem., 42 (1978) 1661. D. Cordier, R. Couturier, L. Grasset and A. Ville, Enzyme Microb. Technol., 4 (1982) 249. A. Kuzuhara, T. Asakura, R. Tomoda and T. Matsunaga, J.Biotechnd., 5 (1987) 199. T. Asakura, Bioindustry, 4 (1987) 36. T. Asakura, H. Yoshimizu, A. Kuzuhara and T. Matsunaga, J. Seric. Sci. Jpn., 57 (1988) 203. T. Asakura, J. Kanetake and M. Demura, J. Polym. Plast. Technol. Eng., 28 (1989) 453. M. Demura and T. Asakura, Biotechnol. Bioeng., 33 (1989) 598. M. Demura, T. Asakura, E. Nakamura and H. Tamura, J. Biotechnol., 10 (1989) 113. M. Demura, T. Asakura and T. Kuroo, Biosensors, 4 (1989) 361. H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 127. H. Yoshimizu and T. Asakura, J. Appl. Polym. Sci., 40 (1990) 1745. T. Asakura, H. Yoshimizu and M. Kakizaki, Biotechnol. Bioeng., 35 (1990) 511. R. Tor and A. Freeman, Anal. Chem., 58 (1979) 1042. T. Shinbo, M. Sugiura and N. Kamo, Anal. Chem., 51 (1979) 100. R.H. William and H.B. HalsaIl, Anal. Chem., 57 (1985) 1321A. A. Friboulet and D. Thomas, Biophys. Chem., 16 (1982) 153. M. Sugiura, Nippon Nogei Kagaku Kaishi, 47 (1973) 563. M. Demura, A. Kitamura, A. Shibamoto and T. Asakura, J. Appl. Polym. Sci., 36 (1988) 535. T. Asakura, Y. Watanabe, A. Uchida and H. Minagawa, Macromolecules, 17 (1984) 1075. A. David, M. Metayer, D. Thomas and G. Broum, J. Membrane Biol., 18 (1974) 113. T. Ciftci and W.R. Vieth, J. Mol. Catal., 8 (1980) 455. Y. Kobatake, K. Kurihara N. Kamo in T. Takamura and A. Kozawa (Eds.), Surface Electrochemistry. Advances and Concepts, Japan Scientific Societies Press, Tokyo, 1978, p. 1.