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A specific enzyme electrode for l -glutamate-development and application
Biosensors 4 (1989) 381-391
A Specific Enzyme Electrode for L-glutamateDevelopment and Application
Ulla Wollenberger, Frieder W. Scheller, Anette Bdhmer, Margrit Passarge & Hans-Georg Miiller Central Institute of Molecular Biology, Academy of Sciences of the GDR, Robert-RossleStrasse lo,1115 Berlin-Buch, GDR (Received 16 June 1988; revised version received and accepted 24 February 1989)
ABSTRACT A biosensor for the specific determination of &.&amate has been developed using L-glutamate oxidase in combination with a hydrogen peroxide indicating electrode. The biosensor response depends linearly on L-glutamate concentrau’on between 0401 and l-0 mM. The measuring time is 2 min. The sensor is stable for more than 10 days during which more than 500 assays can be performed. The sensor has been applied to L-glutamate determination in liquid seasonings. Furthermore, transaminase activities have been determined by their catalytic L-glutamate production from alpha-ketoglutarate and L-alanine or L-aspartate. Also, the coimmobilization of glutaminase yielded a bienzyme electrode sensitive to L-glutamine. Key words: glutamate sensor, glutamate oxidase, alanine ferase, aspartate aminotransferase, glutamine sensor.
aminotrans-
INTRODUCTION The determination of L-glutamate (glutamate) is important for the control of fermentation in the foodstuff industry, since it is contained in many kinds of foods as an essential flavour compound. Furthermore, glutamate produced in enzyme reactions, e.g. by alanine aminotransferase and aspartate aminotransferase, can be a measure of the respective enzyme 381 Biosensors 0265-928X/89/$03.50 @ 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
Ulla Wollenberger et al.
382
activity. These data are important in the diagnosis of heart and liver diseases. Several biospecific electrodes for glutamate have been proposed using immobilized glutamine synthetase (Iida et al., 1986), glutamate decarboxylase (Ahn et al., 1975), glutamate dehydrogenase (Malinauskas & Kulys, 1978) and glutamate oxidase (Yamauchi et aE., 1983), which were either sensitive or unstable to varying degrees. The principle of enzymatic substrate recycling for highly sensitive glutamate determination has been studied by Schubert et al. (1986) using glutamate dehydrogenase and alanine aminotransferase. However, this sensor was quite unstable. In addition, plant tissue slices (Kuriyama & Rechnitz, 1981) and whole microorganisms (Hikuma et aE., 1980; Riedel & Scheller, 1987) have been used which are, however, intrinsically unspecific and slow responding. Recently, a new highly specific FAD-enzyme, glutamate oxidase was isolated (BBhmer et aE., in press), catalysing the following reaction: L-glutamate + O2 + Hz0 --+ alpha-ketoglutarate + NH3 + H202
(1)
This paper describes the construction and application of an enzyme electrode for the determination of glutamate using this new glutamate oxidase.
EXPERIMENTAL Materials
Glutamate
oxidase (GLOD) was isolated and purified from a culture of Streptumyces endus as described by Biihmer et al. (in press). The specific activity of the enzyme preparation was l-2 Ulmg protein. Asparaginase (80 Ulmg), including a glutaminase activity, was purchased from Boeringer Mannheim. Photogelatin was obtained from VEB Gelatinewerk Calbe, GDR. The dialysis membrane (cellulose) was obtained from VEB CK Bitterfeld, GDR. All other reagents used were of analytical grade. Apparatus preparation
and
pr~~ure~nzyme
immob~l~ation
and
electrode
GLOD membranes were prepared by gelatin entrapment (Scheller et al., 1979). Aliquots of GLOD or mixtures of GLOD and human serum ~bumin with a protein content of 1.2 mglml were added to 200 ~1 of 5% gelatin solution. The resulting solutions were cast on a 4 cm2 area of a plane polyethylene support and dried at 4°C for at least 12 h. The
A specific enzyme electrode for L-glutamate
383
membrane was then removed from the support and dry stored at 4°C until use. For the preparation of the sensor, a 3 mm X 3 mm piece of enzyme layer was sandwiched between two dialysis membranes and attached to the hydrogen peroxide sensitive electrode (Pt-Ag/AgCl/O-1 M KCI). For the glutamine electrode 5 ~1 asparaginase were dropped onto the piece of GLOD layer. With this membrane the electrode was prepared in analogy to the glutamate sensor. The enzyme electrode was mounted in a thermostatically controlled measuring cell and connected to a polarograph (GWP 673, ZWG Berlin, GDR) or used as a part of the enzyme electrode based analyser Glukometer (ZWG Berlin, GDR). The measuring cell was equipped with a magnetic stirrer and contained 2 ml of the appropriate buffer. After sample addition either the current-time curve or its first derivative was recorded on a chart recorder (endim 620.01, VEB MeSgeratewerk Schlotheim, GDR). The Glukometer device displays the maximum of the first derivative of the current-time curve. For flow-through measurements, the Glukometer was combined with a flow-through cell, an automatic sampler APS 4 and a peristaltic pump DP 2-2 (VEB MLW, Medingen, GDR) and a chart recorder. The flow rate was O-8 ml/min. For transaminase measurements, the current-time curve was recorded.
RESULTS AND DISCUSSION Characterization
of the glutamate sensor
The response of the biosensor to glutamate was optimized with respect to pH and buffer solution. The sensitivity with Tris buffer (O-05 and O-1 M) was about 40% of that in phosphate (Sorensen) or phosphate-citrate (McIlvaine) buffer. A slight decrease in sensitivity was observed with increasing ionic strength of the buffer solution above O-1 M. Therefore, Sorensen phosphate buffer (O-066 M) was chosen for all further investigations. The pH profile of the glutamate measurements shows an optimum pH range of 7-0-7.2 (Fig. 1). To study various effecters, the enzyme solution (30 mu/ml) and the enzyme electrode were preincubated with 1 mM of the particular substance for 5 min before addition of 5 mM glutamate. The residual activities determined are summarized in Table 1. GLOD was irreversibly inhibited by Ag+ and Hg2+ ions. A slight inhibition was observed with PCMB, KCN and NaN,, whereas metal chelating agents had no effect on the enzyme activity. Thus SH-groups may be involved in the catalytic activity of the
384
Ulla Wollenberger et al.
Fig. 1. Effect of pH on the response of the glutamate electrode obtained in McIlvaine buffer after addition of 0.5 mM (0) or 1-OmM (0) L-glutamate concentration at T = 25°C.
enzyme. Similar results have been described for GLOD from Streptomyit is obvious that the immobilization of the enzyme prevents some of the inhibition effects observed with the soluble enzyme. Furthermore, no activator was found. To study the specificity of the biosensor, the influence of various amino acids and a number of possible interfering substances present in biological samples was investigated, including creatinine, pyruvate, lactate and glucose. In agreement with the data obtained by photometric methods (Bohmer et al., in press), glutamate serves as an exclusive substrate. On the other hand, GLOD from Streptomyces viohcens oxidizes r_-glutamine and L-histidine (Kamei et al., 1983). Alpha-ketoglutarate, a transaminase substrate, was recognized to be a product inhibitor of GLOD, even when the enzyme was immobilized. In practical use for glutamate measurement, this influence should be negligible. However, in monitoring transaminase activities for clinical analysis, this sensor works with a diminished sensitivity. Since the concentration change of alpha-ketoglutarate during the transaminase catalysed reaction is small, the influence on sensitivity is almost constant. Interference may also occur at the electrochemical indicator reaction level. Ascorbic acid, for instance, which is anodically oxidized at +600 mV, is indicated with a sensitivity 45% higher than that for glutamate. Usually, its concentration in the sample is small compared to glutamate. ces viokzscens (Kamei et al., 1983). Furthermore,
385
A specific enzyme electrode for L-glutamate TABLE 1
Influence of Effecters on Soluble GLOD and GLOD electrode Response at Addition of 5 mM Glutamate Relative activity (%) Effector (1 mM) PCMB” DTNBb 44’Bipyridine PMSF EDTAd KCN NaN3 AgNQ Ca(NWz f-fgCl2 FAD alpha KG’
Soluble GLOD 100 82 94 92 80 100 58 87 6 94 0
GLOD electrode 100 100 100 100 100 68 92 44 irreversible 97 0 irreversible 100 89
“p-chlormercuribenzoic acid. b2 2’dinitrod,5’-dithiodibenzoic acid. cphenylmethanesulphonylfluoride. dethylene-diaminetetraacetic acid. ‘alpha-ketoglutarate.
The influence of enzyme loading on the current signal was investigated between 0.01 and O-6 U/cm*. The upper value was set by the low activity of of the enzyme preparation used. A constant protein concentration 0.12 mg/cm* was maintained by substituting GLOD with the corresponding amount of human serum albumin to ensure a constant thickness of the membrane. As seen in Fig. 2, the sensitivity levels off at about O-3 U/cm*. Increasing the stirring speed above 60 r.p.m. did not increase the sensitivity. These results indicate that the biosensor is controlled by internal mass transport. The enzyme activity remaining after immobilization was about 51%. For all further measurements GLOD membranes with an activity of O-5 U/cm2 were used. Figure 3 shows typical response curves of the enzyme electrode to glutamate. In the steady state method, addition of glutamate causes a rapid current increase due to the hydrogen peroxide production in the enzyme layer, which reaches a steady state within 40 s. The measuring time is about 5 min. A considerable reduction of measuring time to less
386
Ulla Wollenberger et al.
Fig. 2. Effect of enzyme loading on the response of the glutamate electrode; 0.12 mgkm* protein content, O-2 mh4 glutamate concentration.
0.2
0.4
0.6
0.8
I-glutamateCmM
1.0 1
Fig. 3. Typical response curves of the glutamate electrode obtained in the stationary state mode; O-5 U/cm* GLOD, pH 7.2 phosphate buffer.
than 2 min is reached using the kinetic current-time curve method. In this mode, the coefficient of variation is below 2% for a sample containing 2-O and 20.0 mM glutamate. The sensor has a useful lifetime of 10 days (Fig. 4), during which more than 500 assays can be performed. The calibration graph (Fig. 5) shows a linear concentration range of O@Ol-1-OmM glutamate (r = O-998). Using the flow-through analyser, linearity was obtained up to 0.8 mM glutamate (r = 0.999). In contrast to the results obtained with the manual Glukometer device, the lower detection limit was only 0.010 mM glutamate. The measuring frequency using the flow-through system was as high as 60/h, and the relative standard deviation of the method using O-2 mM glutamate (8 mM in the sample) was usually below
387
A specific enzyme’ electrode for L-glutamate
6
la
5 the
20
tdl
Fig. 4. Long-term stability of a glutamate electrode, 0.5 U/cm2 GLOD in gelatin, 0*2 mM (0) and O-8 m&i (0) glutamate concentration.
50
T R
25
0.5
1.0 L-glutamate
Fig. 5. Dependence
1.5
2.0
CmMI
of the current of the glutamate electrode on glutamate ~n~ntration; O-5 U/cm2 GLOR, pH 7-2 phosphate buffer.
388
Vlla Wollenberger et al.
2% (n = 20). It is known that the sensitivity of a diffusion controlled enzyme electrode is inversely proportional to the thickness of the membrane (Carr & Bowers, 1980). The configuration of the enzyme electrodes used in the Glukometer device results in a lower membrane thickness. Therefore, the obtained differences in detection limits are plausible. When the sensor was stored overnight at 4”C, a stability of at least 10 days was also obtained with the flow-through electrode. Application of the glutamate electrode The sensor was used in the determination of glutamate in serum and liquid seasonings. For recovery studies, glutamate (4-20 mM) was added to these samples and the concentration of glutamate was determined in triplicate with the Glukometer. As shown in Table 2, a recovery between 89 and 106% with an average of 103.7 + 3% and 96.1 f 6% for diluted standard serum and liquid seasoning, respectively, were obtained. Thus the sensor is applicable for determination of glutamate in foodstuff and serum. Determination of transaminases activity with the glutamate electrode In order to measure the activity of alanine aminotransferase and aspartate aminotransferase, the glutamate, pyruvate, and oxaloacetate production catalysed by these enzymes can be followed: alanine aminotransferase: L-alanine + alpha-ketoglutarate G aspartate aminotransferase: L-aspartate + alpha-ketoglutarate G
L-glutamate + pyruvate L-glutamate + oxaloacetate
(2) (3)
Recently, a few biosensors for the determination of transaminase activity have been proposed. These include pyruvate sensitive monoenzyme (Mizutani et al., 1980) and bienzyme (Schubert et al., 1986) electrodes. However, they can only be applied to alanine aminotransferase assay. Kihara et al. (1984) applied a bienzyme electrode, comprising oxaloacetate decarboxylase and pyruvate oxidase adsorbed to polyvinyl chloride, for the sequential measurement of both transaminases. The detection of glutamate as a common product of both transaminases allows the determination of both enzymes with a monoenzyme electrode. We applied the developed glutamate sensor to transaminase activity measurement. After addition of a 200 ~1 sample to l-8 ml substrate solution, consisting of 0.066 M Siirensen phosphate buffer, pH7.4, 100 mM aspartate or
A specific enzyme electrode for L-glutamate
389
TABLE2 Recovery of L-glutamate Added to Diluted Standard Serum and Diluted Liquid Seasonings Obtained with the Glukometer Device Sample
Added
Standard serum (O-25rnM)
(m,u)
4 10 20 4 10 20 4 10 20
Liquid seasoning (4.97 rnM)
Liquid seasoning (4.11 rnM)
Found (mM)
Recovery (%)
4.48 + O-04 10.84 f O-36 20.12 + 0.50 8.58 If;o-57 15.10 f 0.05 22.87 1-064 7.67 f 0.45 14.20 t- 0.41 24.51+- 0.22
105.9 + 0.8 106*0& 3.3 99.3 k 2.5 90-3 a 6.7 101.2 Z!Z 0.3 89.4 + 2.8 92.8 + 5.9 101.2 + 2.9 101.9 + 0.9
and 5 mM alpha-ketoglutarate, a current increase was observed. It reflects glutamate formation by the reaction catalysed by aspartate aminotransferase or alanine aminotransferase. From the slope of the linear current increase and glutamate calibration graphs, the respective enzyme activity can be calculated. The slope depends linearly on transaminase activity between 2 and 200 U/litre for both alanine aminotransferase (Fig. 6) and aspartate aminotransferase. This corresponds to 20-2000 U/litre in the sample. Therefore, it is considered that the enzyme electrode can be used for the determination of transaminase activities in serum samples. A detailed description is in preparation.
225 mM alanine
Glutamine electrode The application of additional immobilized asparaginase, with glutaminase activity as quoted by the producer (Boehringer Mannheim), yielded a bienzyme electrode for glutamine. Here glutamate produced in the glutaminase catalysed reaction (Eqn 4) is sequentially oxidized in the GLOD layer. The resulting anodic current increase is a measure of the glutamine/ glutamate content of the sample. L-glutamine + H20+r_-glutamate +NHs The conditions for glutamate determination zyme sensor. A linear current-concentration
(4)
are the same for this bien-
dependence was obtained for O-1-1-8 mM glutamine (Fig. 7). However, the sensitivity was only 5% compared with glutamate. The measuring time was 4 min. Owing to the low glutaminase activity, the resulting sensor had a life time of only two
Vlla Wollenberger et al.
390
10
20
OPT l3wflt
Fig. 6. Dependence
I
40
30
50
3
of the slope of the current-time curve on activity of alanine aminotransferase (GPT).
-p 01
I-glutomate 0.2
! z
I
.---* .
40
./
22 I7
2.0
/
/’ ~ :/ i
-*-
20 1.0 I-gtutamme CmM3
Fig. 7. Current dependence of the glutamine electrode on glutamate and glutamine concentrations; 0.5 U/cm2 GLOD and 0.05 U/cm’ glutaminase, pH 7.2 phosphate buffer.
A specific enzyme electrodefor L-glutamate
391
days. The dependence on glutamine concentration as well as measuring time corresponds to that described recently by Romette and Cooney (1987).
REFERENCES Ahn, B. K., Wolfson, Jr, S. K. & Yao, S. J. (1975). An enzyme electrode for the determination of r_-glutamic acid. gi~eIe~tro~hern. Biuenergy, 2, 142-53. Bohmer, A., Mtiller, A., Passarge, M., Liebs, P., Honeck, H. & Miiller, H.-G. (1989). A novel L-glutamate oxidase from Streptomyces endus: Purification and properties, Eur. J. Biochem., in press. Carr, P. W. & Bowers, L. D. (1980). Immobilized enzymes in analytical and clini&a~chemistry. John Wiley & Sons, New York. Hikuma, M., Gbana, H., Yasuda, T., Karube, I. & Suzuki, S. (1980). A potentiometric microbial sensor based on immobilized Escherichia coli for glutamic acid. Anal. Chim. Acta, 116,61-7. Iida, T., Kawabe, T., Hisatomi & Mitamura, T. (1986). A long-lifetime Lglutamic acid sensor using glutamine synthetase from a thermophilic bacterium. In Proceedings uf the 2nd International Meeting on Chemical Sensors. Bordeaux, pp. 592-S. Kamei, T., Asano, K., Kondo, H., Matsuzaki, M. & Nakamura, S. (1983). L-glutamate oxidase from Streptomyces violascens, II, Properties. Chem. Pharm. Bull., 31,3609-16. Kihara, K., Yasukawa, E. & Hirose, S. (1984). Sequential determination of glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase activities in serum using an immobilized bienzyme-~Iy~~nyl chloride) membrane electrode. Anal. Chem., 56,187&80. Ruriyama, S. & Rechnitz, G. A. (1981). Plant tissue-based bioselective membrane electrode for glutamate. Anal. Chim. Actu, 131, 91-6. Malinauskas, A. & Kulys, J. J. (1978). Alcohol, lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide. Anal. Chim. Acta, 98,31-7. Mizutani, F., Tsuda, K., Karube, I., Suzuki, S. & Matsumoto, K. (1980). Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor. Anal. Chim. Actu, 118, 65-71. Riedel, K. & Scheller, F. (1987). Inhibitor-treated microbial sensor for the selective determination of glutamic acid. Analyst, 112, 341-2. Romette, J. L. & Cooney, C. L. (1987). L-glutamine enzyme electrode for on-line mammalian cell culture process control. Anal. Lett., 20, 1069-81. Scheller, F., Pfeiffer, D., Jtinchen, M., Seyer, I., Siepe, M. & Pittelkow, R. (1979). GDR patent 127843. Schubert, F., Kirstein, D., Scheller, F., Appelqvist, R., Gorton, L. & Johannsson, G. (1986). Enzyme electrodes for L-glutamate using chemical redox mediators and enzymatic substrate amplification. Anal. Lett., 19, 1273438. Yamauchi, H., Kusakabe, H., Mido~kawa, Y., Fujishima, T. & Kuninaka, A. (1983). Enzyme electrode for specific determination of L-glutamate. In Proceedings of the Biotechnology Congress. Miinchen, 705-10.
A Specific Enzyme Electrode for L-glutamateDevelopment and Application
Ulla Wollenberger, Frieder W. Scheller, Anette Bdhmer, Margrit Passarge & Hans-Georg Miiller Central Institute of Molecular Biology, Academy of Sciences of the GDR, Robert-RossleStrasse lo,1115 Berlin-Buch, GDR (Received 16 June 1988; revised version received and accepted 24 February 1989)
ABSTRACT A biosensor for the specific determination of &.&amate has been developed using L-glutamate oxidase in combination with a hydrogen peroxide indicating electrode. The biosensor response depends linearly on L-glutamate concentrau’on between 0401 and l-0 mM. The measuring time is 2 min. The sensor is stable for more than 10 days during which more than 500 assays can be performed. The sensor has been applied to L-glutamate determination in liquid seasonings. Furthermore, transaminase activities have been determined by their catalytic L-glutamate production from alpha-ketoglutarate and L-alanine or L-aspartate. Also, the coimmobilization of glutaminase yielded a bienzyme electrode sensitive to L-glutamine. Key words: glutamate sensor, glutamate oxidase, alanine ferase, aspartate aminotransferase, glutamine sensor.
aminotrans-
INTRODUCTION The determination of L-glutamate (glutamate) is important for the control of fermentation in the foodstuff industry, since it is contained in many kinds of foods as an essential flavour compound. Furthermore, glutamate produced in enzyme reactions, e.g. by alanine aminotransferase and aspartate aminotransferase, can be a measure of the respective enzyme 381 Biosensors 0265-928X/89/$03.50 @ 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
Ulla Wollenberger et al.
382
activity. These data are important in the diagnosis of heart and liver diseases. Several biospecific electrodes for glutamate have been proposed using immobilized glutamine synthetase (Iida et al., 1986), glutamate decarboxylase (Ahn et al., 1975), glutamate dehydrogenase (Malinauskas & Kulys, 1978) and glutamate oxidase (Yamauchi et aE., 1983), which were either sensitive or unstable to varying degrees. The principle of enzymatic substrate recycling for highly sensitive glutamate determination has been studied by Schubert et al. (1986) using glutamate dehydrogenase and alanine aminotransferase. However, this sensor was quite unstable. In addition, plant tissue slices (Kuriyama & Rechnitz, 1981) and whole microorganisms (Hikuma et aE., 1980; Riedel & Scheller, 1987) have been used which are, however, intrinsically unspecific and slow responding. Recently, a new highly specific FAD-enzyme, glutamate oxidase was isolated (BBhmer et aE., in press), catalysing the following reaction: L-glutamate + O2 + Hz0 --+ alpha-ketoglutarate + NH3 + H202
(1)
This paper describes the construction and application of an enzyme electrode for the determination of glutamate using this new glutamate oxidase.
EXPERIMENTAL Materials
Glutamate
oxidase (GLOD) was isolated and purified from a culture of Streptumyces endus as described by Biihmer et al. (in press). The specific activity of the enzyme preparation was l-2 Ulmg protein. Asparaginase (80 Ulmg), including a glutaminase activity, was purchased from Boeringer Mannheim. Photogelatin was obtained from VEB Gelatinewerk Calbe, GDR. The dialysis membrane (cellulose) was obtained from VEB CK Bitterfeld, GDR. All other reagents used were of analytical grade. Apparatus preparation
and
pr~~ure~nzyme
immob~l~ation
and
electrode
GLOD membranes were prepared by gelatin entrapment (Scheller et al., 1979). Aliquots of GLOD or mixtures of GLOD and human serum ~bumin with a protein content of 1.2 mglml were added to 200 ~1 of 5% gelatin solution. The resulting solutions were cast on a 4 cm2 area of a plane polyethylene support and dried at 4°C for at least 12 h. The
A specific enzyme electrode for L-glutamate
383
membrane was then removed from the support and dry stored at 4°C until use. For the preparation of the sensor, a 3 mm X 3 mm piece of enzyme layer was sandwiched between two dialysis membranes and attached to the hydrogen peroxide sensitive electrode (Pt-Ag/AgCl/O-1 M KCI). For the glutamine electrode 5 ~1 asparaginase were dropped onto the piece of GLOD layer. With this membrane the electrode was prepared in analogy to the glutamate sensor. The enzyme electrode was mounted in a thermostatically controlled measuring cell and connected to a polarograph (GWP 673, ZWG Berlin, GDR) or used as a part of the enzyme electrode based analyser Glukometer (ZWG Berlin, GDR). The measuring cell was equipped with a magnetic stirrer and contained 2 ml of the appropriate buffer. After sample addition either the current-time curve or its first derivative was recorded on a chart recorder (endim 620.01, VEB MeSgeratewerk Schlotheim, GDR). The Glukometer device displays the maximum of the first derivative of the current-time curve. For flow-through measurements, the Glukometer was combined with a flow-through cell, an automatic sampler APS 4 and a peristaltic pump DP 2-2 (VEB MLW, Medingen, GDR) and a chart recorder. The flow rate was O-8 ml/min. For transaminase measurements, the current-time curve was recorded.
RESULTS AND DISCUSSION Characterization
of the glutamate sensor
The response of the biosensor to glutamate was optimized with respect to pH and buffer solution. The sensitivity with Tris buffer (O-05 and O-1 M) was about 40% of that in phosphate (Sorensen) or phosphate-citrate (McIlvaine) buffer. A slight decrease in sensitivity was observed with increasing ionic strength of the buffer solution above O-1 M. Therefore, Sorensen phosphate buffer (O-066 M) was chosen for all further investigations. The pH profile of the glutamate measurements shows an optimum pH range of 7-0-7.2 (Fig. 1). To study various effecters, the enzyme solution (30 mu/ml) and the enzyme electrode were preincubated with 1 mM of the particular substance for 5 min before addition of 5 mM glutamate. The residual activities determined are summarized in Table 1. GLOD was irreversibly inhibited by Ag+ and Hg2+ ions. A slight inhibition was observed with PCMB, KCN and NaN,, whereas metal chelating agents had no effect on the enzyme activity. Thus SH-groups may be involved in the catalytic activity of the
384
Ulla Wollenberger et al.
Fig. 1. Effect of pH on the response of the glutamate electrode obtained in McIlvaine buffer after addition of 0.5 mM (0) or 1-OmM (0) L-glutamate concentration at T = 25°C.
enzyme. Similar results have been described for GLOD from Streptomyit is obvious that the immobilization of the enzyme prevents some of the inhibition effects observed with the soluble enzyme. Furthermore, no activator was found. To study the specificity of the biosensor, the influence of various amino acids and a number of possible interfering substances present in biological samples was investigated, including creatinine, pyruvate, lactate and glucose. In agreement with the data obtained by photometric methods (Bohmer et al., in press), glutamate serves as an exclusive substrate. On the other hand, GLOD from Streptomyces viohcens oxidizes r_-glutamine and L-histidine (Kamei et al., 1983). Alpha-ketoglutarate, a transaminase substrate, was recognized to be a product inhibitor of GLOD, even when the enzyme was immobilized. In practical use for glutamate measurement, this influence should be negligible. However, in monitoring transaminase activities for clinical analysis, this sensor works with a diminished sensitivity. Since the concentration change of alpha-ketoglutarate during the transaminase catalysed reaction is small, the influence on sensitivity is almost constant. Interference may also occur at the electrochemical indicator reaction level. Ascorbic acid, for instance, which is anodically oxidized at +600 mV, is indicated with a sensitivity 45% higher than that for glutamate. Usually, its concentration in the sample is small compared to glutamate. ces viokzscens (Kamei et al., 1983). Furthermore,
385
A specific enzyme electrode for L-glutamate TABLE 1
Influence of Effecters on Soluble GLOD and GLOD electrode Response at Addition of 5 mM Glutamate Relative activity (%) Effector (1 mM) PCMB” DTNBb 44’Bipyridine PMSF EDTAd KCN NaN3 AgNQ Ca(NWz f-fgCl2 FAD alpha KG’
Soluble GLOD 100 82 94 92 80 100 58 87 6 94 0
GLOD electrode 100 100 100 100 100 68 92 44 irreversible 97 0 irreversible 100 89
“p-chlormercuribenzoic acid. b2 2’dinitrod,5’-dithiodibenzoic acid. cphenylmethanesulphonylfluoride. dethylene-diaminetetraacetic acid. ‘alpha-ketoglutarate.
The influence of enzyme loading on the current signal was investigated between 0.01 and O-6 U/cm*. The upper value was set by the low activity of of the enzyme preparation used. A constant protein concentration 0.12 mg/cm* was maintained by substituting GLOD with the corresponding amount of human serum albumin to ensure a constant thickness of the membrane. As seen in Fig. 2, the sensitivity levels off at about O-3 U/cm*. Increasing the stirring speed above 60 r.p.m. did not increase the sensitivity. These results indicate that the biosensor is controlled by internal mass transport. The enzyme activity remaining after immobilization was about 51%. For all further measurements GLOD membranes with an activity of O-5 U/cm2 were used. Figure 3 shows typical response curves of the enzyme electrode to glutamate. In the steady state method, addition of glutamate causes a rapid current increase due to the hydrogen peroxide production in the enzyme layer, which reaches a steady state within 40 s. The measuring time is about 5 min. A considerable reduction of measuring time to less
386
Ulla Wollenberger et al.
Fig. 2. Effect of enzyme loading on the response of the glutamate electrode; 0.12 mgkm* protein content, O-2 mh4 glutamate concentration.
0.2
0.4
0.6
0.8
I-glutamateCmM
1.0 1
Fig. 3. Typical response curves of the glutamate electrode obtained in the stationary state mode; O-5 U/cm* GLOD, pH 7.2 phosphate buffer.
than 2 min is reached using the kinetic current-time curve method. In this mode, the coefficient of variation is below 2% for a sample containing 2-O and 20.0 mM glutamate. The sensor has a useful lifetime of 10 days (Fig. 4), during which more than 500 assays can be performed. The calibration graph (Fig. 5) shows a linear concentration range of O@Ol-1-OmM glutamate (r = O-998). Using the flow-through analyser, linearity was obtained up to 0.8 mM glutamate (r = 0.999). In contrast to the results obtained with the manual Glukometer device, the lower detection limit was only 0.010 mM glutamate. The measuring frequency using the flow-through system was as high as 60/h, and the relative standard deviation of the method using O-2 mM glutamate (8 mM in the sample) was usually below
387
A specific enzyme’ electrode for L-glutamate
6
la
5 the
20
tdl
Fig. 4. Long-term stability of a glutamate electrode, 0.5 U/cm2 GLOD in gelatin, 0*2 mM (0) and O-8 m&i (0) glutamate concentration.
50
T R
25
0.5
1.0 L-glutamate
Fig. 5. Dependence
1.5
2.0
CmMI
of the current of the glutamate electrode on glutamate ~n~ntration; O-5 U/cm2 GLOR, pH 7-2 phosphate buffer.
388
Vlla Wollenberger et al.
2% (n = 20). It is known that the sensitivity of a diffusion controlled enzyme electrode is inversely proportional to the thickness of the membrane (Carr & Bowers, 1980). The configuration of the enzyme electrodes used in the Glukometer device results in a lower membrane thickness. Therefore, the obtained differences in detection limits are plausible. When the sensor was stored overnight at 4”C, a stability of at least 10 days was also obtained with the flow-through electrode. Application of the glutamate electrode The sensor was used in the determination of glutamate in serum and liquid seasonings. For recovery studies, glutamate (4-20 mM) was added to these samples and the concentration of glutamate was determined in triplicate with the Glukometer. As shown in Table 2, a recovery between 89 and 106% with an average of 103.7 + 3% and 96.1 f 6% for diluted standard serum and liquid seasoning, respectively, were obtained. Thus the sensor is applicable for determination of glutamate in foodstuff and serum. Determination of transaminases activity with the glutamate electrode In order to measure the activity of alanine aminotransferase and aspartate aminotransferase, the glutamate, pyruvate, and oxaloacetate production catalysed by these enzymes can be followed: alanine aminotransferase: L-alanine + alpha-ketoglutarate G aspartate aminotransferase: L-aspartate + alpha-ketoglutarate G
L-glutamate + pyruvate L-glutamate + oxaloacetate
(2) (3)
Recently, a few biosensors for the determination of transaminase activity have been proposed. These include pyruvate sensitive monoenzyme (Mizutani et al., 1980) and bienzyme (Schubert et al., 1986) electrodes. However, they can only be applied to alanine aminotransferase assay. Kihara et al. (1984) applied a bienzyme electrode, comprising oxaloacetate decarboxylase and pyruvate oxidase adsorbed to polyvinyl chloride, for the sequential measurement of both transaminases. The detection of glutamate as a common product of both transaminases allows the determination of both enzymes with a monoenzyme electrode. We applied the developed glutamate sensor to transaminase activity measurement. After addition of a 200 ~1 sample to l-8 ml substrate solution, consisting of 0.066 M Siirensen phosphate buffer, pH7.4, 100 mM aspartate or
A specific enzyme electrode for L-glutamate
389
TABLE2 Recovery of L-glutamate Added to Diluted Standard Serum and Diluted Liquid Seasonings Obtained with the Glukometer Device Sample
Added
Standard serum (O-25rnM)
(m,u)
4 10 20 4 10 20 4 10 20
Liquid seasoning (4.97 rnM)
Liquid seasoning (4.11 rnM)
Found (mM)
Recovery (%)
4.48 + O-04 10.84 f O-36 20.12 + 0.50 8.58 If;o-57 15.10 f 0.05 22.87 1-064 7.67 f 0.45 14.20 t- 0.41 24.51+- 0.22
105.9 + 0.8 106*0& 3.3 99.3 k 2.5 90-3 a 6.7 101.2 Z!Z 0.3 89.4 + 2.8 92.8 + 5.9 101.2 + 2.9 101.9 + 0.9
and 5 mM alpha-ketoglutarate, a current increase was observed. It reflects glutamate formation by the reaction catalysed by aspartate aminotransferase or alanine aminotransferase. From the slope of the linear current increase and glutamate calibration graphs, the respective enzyme activity can be calculated. The slope depends linearly on transaminase activity between 2 and 200 U/litre for both alanine aminotransferase (Fig. 6) and aspartate aminotransferase. This corresponds to 20-2000 U/litre in the sample. Therefore, it is considered that the enzyme electrode can be used for the determination of transaminase activities in serum samples. A detailed description is in preparation.
225 mM alanine
Glutamine electrode The application of additional immobilized asparaginase, with glutaminase activity as quoted by the producer (Boehringer Mannheim), yielded a bienzyme electrode for glutamine. Here glutamate produced in the glutaminase catalysed reaction (Eqn 4) is sequentially oxidized in the GLOD layer. The resulting anodic current increase is a measure of the glutamine/ glutamate content of the sample. L-glutamine + H20+r_-glutamate +NHs The conditions for glutamate determination zyme sensor. A linear current-concentration
(4)
are the same for this bien-
dependence was obtained for O-1-1-8 mM glutamine (Fig. 7). However, the sensitivity was only 5% compared with glutamate. The measuring time was 4 min. Owing to the low glutaminase activity, the resulting sensor had a life time of only two
Vlla Wollenberger et al.
390
10
20
OPT l3wflt
Fig. 6. Dependence
I
40
30
50
3
of the slope of the current-time curve on activity of alanine aminotransferase (GPT).
-p 01
I-glutomate 0.2
! z
I
.---* .
40
./
22 I7
2.0
/
/’ ~ :/ i
-*-
20 1.0 I-gtutamme CmM3
Fig. 7. Current dependence of the glutamine electrode on glutamate and glutamine concentrations; 0.5 U/cm2 GLOD and 0.05 U/cm’ glutaminase, pH 7.2 phosphate buffer.
A specific enzyme electrodefor L-glutamate
391
days. The dependence on glutamine concentration as well as measuring time corresponds to that described recently by Romette and Cooney (1987).
REFERENCES Ahn, B. K., Wolfson, Jr, S. K. & Yao, S. J. (1975). An enzyme electrode for the determination of r_-glutamic acid. gi~eIe~tro~hern. Biuenergy, 2, 142-53. Bohmer, A., Mtiller, A., Passarge, M., Liebs, P., Honeck, H. & Miiller, H.-G. (1989). A novel L-glutamate oxidase from Streptomyces endus: Purification and properties, Eur. J. Biochem., in press. Carr, P. W. & Bowers, L. D. (1980). Immobilized enzymes in analytical and clini&a~chemistry. John Wiley & Sons, New York. Hikuma, M., Gbana, H., Yasuda, T., Karube, I. & Suzuki, S. (1980). A potentiometric microbial sensor based on immobilized Escherichia coli for glutamic acid. Anal. Chim. Acta, 116,61-7. Iida, T., Kawabe, T., Hisatomi & Mitamura, T. (1986). A long-lifetime Lglutamic acid sensor using glutamine synthetase from a thermophilic bacterium. In Proceedings uf the 2nd International Meeting on Chemical Sensors. Bordeaux, pp. 592-S. Kamei, T., Asano, K., Kondo, H., Matsuzaki, M. & Nakamura, S. (1983). L-glutamate oxidase from Streptomyces violascens, II, Properties. Chem. Pharm. Bull., 31,3609-16. Kihara, K., Yasukawa, E. & Hirose, S. (1984). Sequential determination of glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase activities in serum using an immobilized bienzyme-~Iy~~nyl chloride) membrane electrode. Anal. Chem., 56,187&80. Ruriyama, S. & Rechnitz, G. A. (1981). Plant tissue-based bioselective membrane electrode for glutamate. Anal. Chim. Actu, 131, 91-6. Malinauskas, A. & Kulys, J. J. (1978). Alcohol, lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide. Anal. Chim. Acta, 98,31-7. Mizutani, F., Tsuda, K., Karube, I., Suzuki, S. & Matsumoto, K. (1980). Determination of glutamate pyruvate transaminase and pyruvate with an amperometric pyruvate oxidase sensor. Anal. Chim. Actu, 118, 65-71. Riedel, K. & Scheller, F. (1987). Inhibitor-treated microbial sensor for the selective determination of glutamic acid. Analyst, 112, 341-2. Romette, J. L. & Cooney, C. L. (1987). L-glutamine enzyme electrode for on-line mammalian cell culture process control. Anal. Lett., 20, 1069-81. Scheller, F., Pfeiffer, D., Jtinchen, M., Seyer, I., Siepe, M. & Pittelkow, R. (1979). GDR patent 127843. Schubert, F., Kirstein, D., Scheller, F., Appelqvist, R., Gorton, L. & Johannsson, G. (1986). Enzyme electrodes for L-glutamate using chemical redox mediators and enzymatic substrate amplification. Anal. Lett., 19, 1273438. Yamauchi, H., Kusakabe, H., Mido~kawa, Y., Fujishima, T. & Kuninaka, A. (1983). Enzyme electrode for specific determination of L-glutamate. In Proceedings of the Biotechnology Congress. Miinchen, 705-10.