ANALYTICAL
BIOCHEMISTRY
201,227-232
(19%)
Determination of Glutamic Acid Decarboxylase and Inhibition by an H,O,-Sensing Glutamic Acid Oxidase Biosensor Claudio Botr;, and Fernando
Francesco Porcelli
Botr&,*,’
Marco
Galli,
Giampiero
Lorenti,
Activity
Franc0
Mazzei,
Dipartimento di Studi di Chimica e Tecnologia de& Sostanze Biologicamente Attive, Universitci “La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy, and *Istituto di Merceologia, Facoltci di Economia e Commercio, Universitci “La Sapienza” Via de1 Castro Laurenziano, g-00161 Rome, Italy
Received
July
8, 1991
The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamateselective biosensor. The biosensor is composed of an amperometric HzOz electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H,Oz produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to a-ketoglutaric acid. Since GA is transformed to y-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental re0 1992 Academic sults and ease, time, and cost of operation. Press,
”
Inc.
Glutamic acid decarboxylase (GAD)2 (EC 4.1.1.15) is a highly specific enzyme, localized in a great variety of tissues of the central nervous system (1,2). Its function in the brain is to control y-aminobutyric acid (GABA) ’ To whom correspondence should be addressed. ’ Abbreviations used: GAD, glutamic acid decarboxylase; y-aminobutyric acid; GA, L-glutamic acid, GAO, glutamic dase; VA, valproic acid. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
GABA, acid oxi-
synthesis, by catalyzing the cY-decarboxylation of L-glutamic acid (GA) (reaction 1):
L-glutamic acid + H,O s y-aminobutyric
acid + CO,
[l]
This reaction is believed to be the rate-limiting step of the overall process that assures steady-state concentrations of GABA in brain and in other tissues (3-6). It follows that measurement of both GAD levels and GAD catalytic activity assumes a relevant significance in a broad range of neurophysiological and neuropharmacological studies (7-9). Apart from immunological techniques, the first intent of which is to find out the total amount, rather than the catalytic activity, of GAD in biological samples (lO,ll), in the main two classes of different procedures are employed for the determination of GAD catalytic activity: according to reaction (l), indeed, a quantitative determination of GAD activity can be performed by monitoring the rate of either CO, or GABA production as well as of GA consumption. As exhaustively reviewed by Holdiness (12), determination of carbon dioxide produced by decarboxylation of GA can be followed by manometric measurements as well as by radiochemical monitoring of 14C0, evolved from L-[“C]GA, while the direct or indirect determination of GABA production, as well as of GA consumption, is usually performed by chromatographic methods. The purpose of our work is to present a simple method for the direct and continuous measurement of the catalytic activity of GAD, without the need for any pretreatment process or sample purification. For this 227
Inc. reserved.
228
BOTRti
ET AL.
When the biosensor, connected to an amperometric detector (ABD, Universal Sensors, Inc.), is dipped in a GA solution, its internal H,O, electrode records the H,O, production that takes place at the level of the biocatalytic GAO membrane, according to the reaction 2 5
L-glutamate
+ 0, + Hz0 s a-ketoglutarate
,/
/O-
4
FIG. 1. Schematic representation of the H,O,-sensing glutamic oxidase biosensor: 1, H,O, electrode; 2, cellulose acetate membrane; 3, GAO membrane; 4, 0 ring; 5, dialysis membrane.
purpose we have followed an electroanalytical approach that is analogous to that already applied in the past for the potentiometric determination of carbonic anhydrase activity (13). The selective electrochemical device employed in the present work is represented by a glutamic acid biosensor, which is composed of an amperometric hydrogen peroxide electrode and a glutamic acid oxidase (GAO) (E.C. 1.4.3.11) membrane. In this way it is possible to monitor the rate of GA consumption, rather than that of CO, or GABA production, under the catalytic action of GAD. MATERIALS
Glutamic
AND
METHODS
Acid Biosensor
The glutamic acid biosensor (Universal Sensors, Inc.) is composed of two major components: (a) an internal amperometric H,O,-sensing electrode and (b) a biocatalytic membrane, fixed on the sensing tip of the electrode by a rubber 0 ring, containing an adequate amount of immobilized GAO (0.137 mg/cm’) (Fig. 1). This latter component has also been prepared in our laboratory, by physicochemical immobilization of GAO, from Streptomyces sp. X-119-6 (Yamasa Shoyu Co., Ltd., Tokyo, Japan) on carboxylic membranes (“Biodyne,” Pall Italia SpA, Milano, Italy), by means of the prepolymer of polyazetidine (“Hercules Polycup 172,” Hercules, Inc., Wilmington, DE), according to a procedure already described and discussed (14).
[2]
It follows that there is a direct relationship between the concentration of GA in the sample and the amount of hydrogen peroxide produced at the level of the electrode biomembrane. Enzymatic
3
+ NH, + H20,
Experiments
Amperometric measurements of GAD catalytic activity were carried out by monitoring the speed of GA consumption in the presence of GAD. All experiments were performed at 37 f O.l”C, in a 0.1 M acetate buffer at pH 5.000, in a lo-ml thermostated bath, under constant magnetic stirring. Glutamic acid decarboxylase (19.7 U/mg solid) and L-glutamic acid were both supplied by Sigma (Cat. No. G-3757 and No. G-1251 respectively). Valproic acid (VA) was kindly supplied by Sigma Tau SpA (Pomezia, Rome, Italy). All chemicals were analytical grade. Doubly distilled water was used for the preparation of all reagents solutions. RESULTS
Characterization
of the Glutamic
Acid Biosensor
The equations of the calibration curves, performed at different pH and in two different buffer solutions (0.1 M Tris/HCl, pH 7.500, and 0.1 M acetate, pH 5.000), and the main electroanalytical properties of the GA biosensor are shown in Table 1. The two different experimental conditions refer to the optimum operating conditions of the GAO electrode (Tris/HCl buffer, pH 7.500) (14) and to the experimental conditions imposed by the study of GAD activity (acetate buffer, pH 5.000). Electrode characterization was also performed in the presence of 0.12 M valproic acid: no significant alteration of electrode performance was detected. Enzymatic
Activity
Experiments
The catalytic activity of GAD has been evaluated by calculating the speed of GA consumption taking place in a buffered GA solution, both in the presence and in the absence of enzyme. Figure 2 shows an example of a complete set of our amperometric experiments. Curves a, b,
AMPEROMETRIC
DETERMINATION
OF GLUTAMIC TABLE
Analytical
37 7.500 Tris/HCl, 0.1 M
BIOCHEMISTRY
201,227-232
(19%)
Determination of Glutamic Acid Decarboxylase and Inhibition by an H,O,-Sensing Glutamic Acid Oxidase Biosensor Claudio Botr;, and Fernando
Francesco Porcelli
Botr&,*,’
Marco
Galli,
Giampiero
Lorenti,
Activity
Franc0
Mazzei,
Dipartimento di Studi di Chimica e Tecnologia de& Sostanze Biologicamente Attive, Universitci “La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy, and *Istituto di Merceologia, Facoltci di Economia e Commercio, Universitci “La Sapienza” Via de1 Castro Laurenziano, g-00161 Rome, Italy
Received
July
8, 1991
The catalytic activity of the enzyme L-glutamic acid decarboxylase (GAD) is determined by an amperometric method based on a recently developed glutamateselective biosensor. The biosensor is composed of an amperometric HzOz electrode and a biocatalytic membrane containing the enzyme glutamic acid oxidase (GAO). The biosensor allows the direct and continuous measurement of GA levels by monitoring the H,Oz produced at the electrode interface as a coproduct of the GAO-catalyzed GA oxidation to a-ketoglutaric acid. Since GA is transformed to y-aminobutyric acid and CO2 under the catalytic activity of GAD, the rate of GA consumption in solution, monitored by the GAO biosensor, represents a reliable measure of GAD catalytic activity. Additional experiments performed in the presence of different concentrations of the GAD inhibitor valproic acid have shown the suitability of the proposed approach for the study of GAD inhibitors also. Discussion of the main experimental characteristics of this new analytical method is given in terms of sensitivity, reproducibility, and reliability of the experimental re0 1992 Academic sults and ease, time, and cost of operation. Press,
”
Inc.
Glutamic acid decarboxylase (GAD)2 (EC 4.1.1.15) is a highly specific enzyme, localized in a great variety of tissues of the central nervous system (1,2). Its function in the brain is to control y-aminobutyric acid (GABA) ’ To whom correspondence should be addressed. ’ Abbreviations used: GAD, glutamic acid decarboxylase; y-aminobutyric acid; GA, L-glutamic acid, GAO, glutamic dase; VA, valproic acid. 0003-2697/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form
GABA, acid oxi-
synthesis, by catalyzing the cY-decarboxylation of L-glutamic acid (GA) (reaction 1):
L-glutamic acid + H,O s y-aminobutyric
acid + CO,
[l]
This reaction is believed to be the rate-limiting step of the overall process that assures steady-state concentrations of GABA in brain and in other tissues (3-6). It follows that measurement of both GAD levels and GAD catalytic activity assumes a relevant significance in a broad range of neurophysiological and neuropharmacological studies (7-9). Apart from immunological techniques, the first intent of which is to find out the total amount, rather than the catalytic activity, of GAD in biological samples (lO,ll), in the main two classes of different procedures are employed for the determination of GAD catalytic activity: according to reaction (l), indeed, a quantitative determination of GAD activity can be performed by monitoring the rate of either CO, or GABA production as well as of GA consumption. As exhaustively reviewed by Holdiness (12), determination of carbon dioxide produced by decarboxylation of GA can be followed by manometric measurements as well as by radiochemical monitoring of 14C0, evolved from L-[“C]GA, while the direct or indirect determination of GABA production, as well as of GA consumption, is usually performed by chromatographic methods. The purpose of our work is to present a simple method for the direct and continuous measurement of the catalytic activity of GAD, without the need for any pretreatment process or sample purification. For this 227
Inc. reserved.
228
BOTRti
ET AL.
When the biosensor, connected to an amperometric detector (ABD, Universal Sensors, Inc.), is dipped in a GA solution, its internal H,O, electrode records the H,O, production that takes place at the level of the biocatalytic GAO membrane, according to the reaction 2 5
L-glutamate
+ 0, + Hz0 s a-ketoglutarate
,/
/O-
4
FIG. 1. Schematic representation of the H,O,-sensing glutamic oxidase biosensor: 1, H,O, electrode; 2, cellulose acetate membrane; 3, GAO membrane; 4, 0 ring; 5, dialysis membrane.
purpose we have followed an electroanalytical approach that is analogous to that already applied in the past for the potentiometric determination of carbonic anhydrase activity (13). The selective electrochemical device employed in the present work is represented by a glutamic acid biosensor, which is composed of an amperometric hydrogen peroxide electrode and a glutamic acid oxidase (GAO) (E.C. 1.4.3.11) membrane. In this way it is possible to monitor the rate of GA consumption, rather than that of CO, or GABA production, under the catalytic action of GAD. MATERIALS
Glutamic
AND
METHODS
Acid Biosensor
The glutamic acid biosensor (Universal Sensors, Inc.) is composed of two major components: (a) an internal amperometric H,O,-sensing electrode and (b) a biocatalytic membrane, fixed on the sensing tip of the electrode by a rubber 0 ring, containing an adequate amount of immobilized GAO (0.137 mg/cm’) (Fig. 1). This latter component has also been prepared in our laboratory, by physicochemical immobilization of GAO, from Streptomyces sp. X-119-6 (Yamasa Shoyu Co., Ltd., Tokyo, Japan) on carboxylic membranes (“Biodyne,” Pall Italia SpA, Milano, Italy), by means of the prepolymer of polyazetidine (“Hercules Polycup 172,” Hercules, Inc., Wilmington, DE), according to a procedure already described and discussed (14).
[2]
It follows that there is a direct relationship between the concentration of GA in the sample and the amount of hydrogen peroxide produced at the level of the electrode biomembrane. Enzymatic
3
+ NH, + H20,
Experiments
Amperometric measurements of GAD catalytic activity were carried out by monitoring the speed of GA consumption in the presence of GAD. All experiments were performed at 37 f O.l”C, in a 0.1 M acetate buffer at pH 5.000, in a lo-ml thermostated bath, under constant magnetic stirring. Glutamic acid decarboxylase (19.7 U/mg solid) and L-glutamic acid were both supplied by Sigma (Cat. No. G-3757 and No. G-1251 respectively). Valproic acid (VA) was kindly supplied by Sigma Tau SpA (Pomezia, Rome, Italy). All chemicals were analytical grade. Doubly distilled water was used for the preparation of all reagents solutions. RESULTS
Characterization
of the Glutamic
Acid Biosensor
The equations of the calibration curves, performed at different pH and in two different buffer solutions (0.1 M Tris/HCl, pH 7.500, and 0.1 M acetate, pH 5.000), and the main electroanalytical properties of the GA biosensor are shown in Table 1. The two different experimental conditions refer to the optimum operating conditions of the GAO electrode (Tris/HCl buffer, pH 7.500) (14) and to the experimental conditions imposed by the study of GAD activity (acetate buffer, pH 5.000). Electrode characterization was also performed in the presence of 0.12 M valproic acid: no significant alteration of electrode performance was detected. Enzymatic
Activity
Experiments
The catalytic activity of GAD has been evaluated by calculating the speed of GA consumption taking place in a buffered GA solution, both in the presence and in the absence of enzyme. Figure 2 shows an example of a complete set of our amperometric experiments. Curves a, b,
AMPEROMETRIC
DETERMINATION
OF GLUTAMIC TABLE
Analytical
37 7.500 Tris/HCl, 0.1 M
