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Development and characterization of a polymer gel with an immobilized enzyme to measure l -glutamate
ANALYTICAL
BIOCHEMISTRY
Development
161, 187-199 (1987)
and Characterization of a Polymer Gel with an Immobilized Enzyme to Measure L-Glutamate
JUANI.KORENBROT,RUTHPERRY,ANDDAVID Departments
of Physiology,
Biochemistry, and Ophthalmology, San Francisco, California
R.~OPENHAGEN University 94143
of California
Medical
School,
Received March 25, 1986 Glutamate dehydrogenase (GDH) is used in an enzyme electrode to measure L-glutamate. GDH is covalently immobilized in a hydrophilic, permeable, and semirigid gel produced by the copolymerization of polyacrylamide and N-acryloxysuccinimide. Experimental conditions necessary to retain GDH in the gel with high efficiency and minimum denaturation are optimized. The abilities of enzymatic cofactors and coenzymes, NADH, NAD, ATP. ADP, GTP, and ZnC&, to protect the enzyme during immobilization are explored. Under optimum experimental procedures an enzyme-containing gel is produced that is reproducible and long lasting in its functional behavior. The gel responds to the presence of L-glutamate with high velocity, the delay being less than 500 ms; high specificity, being lOOO-fold more responsive to L-glutamate than D-ghtaIIIate, D or L-aspartate, and N-acetylhistidine; and high sensitivity, a concentration of about 3 PM can be measured. 0 1987 Academic press, IX. KEY WORDS: immobilized enzyme; enzyme electrode; L-glutamate; glutamate dehydrogenase; neurochemistry; amino acid analysis.
The measurement of the release of neurotransmitter molecules by neurons is an experimental technique that allows the determination of the chemical identity of these molecules and the study of the physiological control of their cellular release. For these measurements, the medium bathing the neuron is subjected to quantitative chemical analysis. Typically, either of two experimental strategies are used. In one method, a relatively large volume of extracellular medium is collected over a period of time and then chemically analyzed. The extracellular medium can be collected in vivo, for example with push-pull cannulaes (1,2), by local dialysis (3,4), or with collecting cups (5), or in vitro, for example by recovering the medium in which neurons are maintained in culture. The second strategy depends on the continuous sampling of the medium in the extracellular space with an appropriate probe placed near the neuron. In in vivo experiments, for
example, polarographic electrodes are placed within the nervous tissue (6,7). In in vitro experiments “bioelectrodes,” constructed by sealing the opening of a “patch” micropipet with a cell membrane fragment responsive to the selected neurotransmitter, are placed near the surface of the neuron (8,9). The continuous chemical sampling of the extracellular medium is the preferred method because it permits the resolution of the kinetics of neurotransmitter release. This experimental advantage, however, can be realized only if the analytical probe in the extracellular space is sufficiently small, specific, and sensitive. These problems can, in principle, be resolved by appropriate design of the probe. The problem of low sensitivity can be overcome by using probes that respond to concentration changes in a design that minimizes extracellular volume so that very small mole changes produce large concentration changes. We have used this strategy, for ex-
187
0003-2697187 $3.00 Copyright0 1987 by Academic All rights of reproduction
Press, Inc. in any form reserved.
188
KORENBROT,
PERRY, AND COPENHAGEN
ample, to measure the small, light-dependent release of Ca ions by single vertebrate rod photoreceptor cells (IO, 11). Because of our interest in rod photoreceptors, a neuron believed to use L-glutamate as a neurotransmitter ( 12), we undertook the development of an experimental system to measure L-glutamate with a concentration-sensitive probe. We elected to develop an enzyme electrode for L-glutamate. Enzyme electrodes are a general class of analytical probes that respond to concentration changes and can be made to be highly sensitive, specific, and small (13-15). In an enzyme electrode, a selected enzyme is physically immobilized in a support placed on or near a physical detector. The molecule to be measured is acted upon by the immobilized enzyme and in this reaction products or cosubstrates, that are readily measured by the physical probe, are produced or consumed in stoichiometric amounts (16). L-Glutamate-sensitive enzyme electrodes based on the activity of either glutamate dehydrogenase ( 17,18), glutamate decarboxylase ( 19-2 1), or glutamate oxidase (22) have been reported previously. Previous available designs, however, proved incompatible with our needs, in particular because we required the ability to produce a microprobe having a diameter less than 50 pm. We describe here an electrode based on the covalent immobilization of glutamate dehydrogenase in a polyacrylamide polymer. We propose that such an electrode, when applied under conditions that minimize extracellular volume ( 10,l l), will allow the study of glutamate release by intact single neurons. This method, therefore, should be of use to those interested in the role of glutamate, a neurotransmitter found throughout the nervous system. More generally, we propose that the rationale of the method should make it applicable to the study of other biologically active molecules secreted by neurons or other cell types for which an enzyme electrode can be designed.
MATERIALS
Materials
AND
METHODS
and Apparatus
Glutamate dehydrogenase (GDH)’ (EC 1.4.1.3) purified from beef liver was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). N-Acryloxysuccinimide (NAS) was synthesized following the method of Adalsteinsson et al. (23). Acrylamide (99.9%), N,N’-methylenebisacrylamide (Bis), ZV,N,N’,N’-tetramethylethylenediamine (TEMED), and ammonium persulfate were obtained from Bio-Rad (Richmond, CA). Pipes (piperazine-N,N’bis(2-ethanesulfonic acid)) was from P-L Biochemicals (Milwaukee, WI), and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Spectrophotometric studies were conducted in a Cary 118C instrument (Varian Instruments, Mountain View, CA). Methods Covalent immobilization of GDH in an acrylamide polymer. The following seven stock solutions were prepared: S 1, 0.65 M Pipes and 0.65 mM EDTA, adjusted to pH 6.5 with NaOH. S2, acrylamide (295 mg/ml) and Bis (6.15 mg/ml) in distilled water. S3, 20 mM NADH in distilled water. S4, TEMED (20% v/v) in distilled water. S5, potassium persulfate in distilled water (25 mg/ml). S6, GDH (10 mg/ml) in 30 mM potassium phosphate buffer at pH 7.0. S7, 20 mM NAS in (CH&SO (dimethyl sulfoxide). Stocks 3-6 were prepared fresh each time. All solutions, except S7, were maintained at 4°C. The solutions listed above are those ’ Abbreviations used: GDH, glutamate dehydrogenase; NAS, N-acryloxysuccinimide; Bis, N,N’methylenebisacrylamide; TEMED, N,N,N’,N’tetramethylenediamine; Pipes, piperazine-N,N’-bis(2ethanesulfonic acid); Hepes, 4-(2-hydroxyethyl)I-piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid: Mops, 4-morpholineethanesulfonic acid.
ENZYME
ELECTRODE
MEASUREMENT
used to produce optimum gels. Enzyme cofactors and activators tested were added in stock S3. Gel production was carried out in a cold room at 4°C. Because the polymerization of acrylamide is inhibited by molecular oxygen we degassed all stock solutions and then saturated them with nitrogen immediately before use. Mixing of stock solutions and gel polymerization were conducted under a nitrogen atmosphere within an enclosed cylindrical Plexiglas chamber ( 14 cm in diameter and 10 cm in height). Degassed solutions Sl-S6 were aliquoted into each of six vials through a small opening in the plastic chamber. Solutions were then transferred from each vial into a seventh, stirred vial in the following volume and sequence: 0.320 ml of S1,0.2 ml of S6, 0.1 ml of S3, and 0.04 ml of S7. Exactly 10 min later 0.273 ml of S2,0.04 ml of S4, and 0.04 ml of S5 were added. When we tested the effects of various chemical constituents and their concentrations on gel production, the stock solutions were adjusted such that the sequence and volume of their addition was unchanged.
Gelfing time and recovery of the polymer. Gelling time was defined as the time interval between the addition of solution S5 and the moment at which the rotation of the stirring bar in the mixing vial ceased. For any given set of experimental conditions this time was reproducible to within + 15%. If gels failed to form within this expected time interval they were discarded. The rare failures could usually be traced to either inadequate removal of oxygen or contamination of stock solutions. Ten minutes after its polymerization the gel was covered in the mixing vial with 0.5 ml of a solution of 0.2 M Hepes (4-(2-hydroxyethyl)1 -piperazineethanesulfonic acid) and 0.2 mM EDTA. Two minutes later the Plexiglas chamber was opened and the gel was transferred with the aid of 25 ml of the Hepes/EDTA solution to the glass vessel of an homogenizer.
OF
L-GLUTAMATE
189
Gel washing and determination of e@ciency of protein retention. The enzyme-containing polymer was thoroughly homogenized at 4°C with a motor-driven, tight-fitting Teflon pestle (A. H. Thomas Co. Philadelphia, PA). The homogenate was mixed with 25 ml of the Hepes/EDTA solution and centrifuged at 4°C for 55 min at 10,OOOg. The supernatant was collected and the gel pellet was washed twice again by resuspension and centrifugation with Hepes/ EDTA solution. The protein content of each of these supernatants was measured colorimetrically using GDH as the standard (Bradford protein assay, Bio-Rad Labs, Richmond CA). The protein content of the gel was determined by subtracting the sum of the protein amounts measured in all supernatants from the protein amount added to the gelforming solution. Efficiency of protein retention in the gel was defined as the ratio of the amount of protein retained by the gel to that added to the gel-forming solution.
Assay of enzymatic activity in the gel. GDH activity was measured spectrophotometrically at room temperature by determining the initial rate of reduction of NAD into NADH. All assays, except those in the stability studies, were completed within 24 h of the production of the gel. Following the last wash and centrifugation, the gel pellet was carefully weighed and resuspended in 0.2 M Hepes, 0.2 mM EDTA, and NAD, pH 8.0, at 0.1 to 0.2 mg gel/ml. The concentration of NAD in this solution was varied in those experiments in which we investigated its effects on enzyme kinetics, otherwise it was 8 mM. The gel suspension was maintained at 4°C until shortly before it was assayed. To assay GDH activity, the gel suspension was vortexed and a 0.5-ml aliquot was rapidly mixed in a quartz cuvette with an equal volume of the Hepes/EDTA/NAD solution containing L-glutamate. The final concentration of L-glutamate in this solution was varied in those experiments in which we investigated its effects on enzyme kinetics,
190
KORENBROT,
PERRY, AND COPENHAGEN
otherwise it was 25 mM. The quartz cuvette was immediately placed in the measuring compartment of a spectrophotometer and its absorbance at 340 nm was measured as a function of time. Enzymatic activity was calculated from the absorbance data given a molar extinction coefficient of NADH at 340 nm of 6.22 X lo6 It cm/M. The specific enzymatic activity in repeat assays of the same gel varied within about 5%; however, this value varied by as much as 20% among different gels produced by the same protocol. In order to average and compare gels produced under varying experimental conditions on different dates we used the following quantitative protocol. The enzyme solution used in the production of each gel (S2 above) was assayed in parallel with the gels and its specific enzymatic activity was thus determined. To normalize and compare data we expressed the specific activity in the gel as a fraction of the specific activity of the enzyme in the solution used to produce that gel. Speed of response of thin gels. To determine the speed of response of the gel to step changes in glutamate concentration we assayed the enzymatic activity of thin, 70 pm, polymer films produced in the device illustrated in Fig. 1. This device could be placed in the measuring compartment of the spectrophotometer and the glutamate concentration in the medium bathing the gel within the device could be changed rapidly. The device consisted of two Plexiglas chambers. In one of them, a round quartz window was mounted with a 70-pm recess relative to the wall of the chamber. A thin film of enzymecontaining polymer was cast in that recess. To do so, this chamber was placed horizontally within the nitrogen-saturated cylindrical chamber described above. The gel-forming solution stocks were mixed as described and 4 min after adding S5, 200 ~1 of the still ungelled solution were placed on top of the quartz window. A heavy block of brass covered with a 3-mm-thick sheath of Teflon was
Inflow
1. Schematic drawing of the chamber used to produce thin GDH-polyacrylamide gels and to measure their enzymatic activity. One Plexiglas chamber (A) had a quartz round window(q) recessed 70 pm relative to the wall of the chamber. The polymer gel was formed within that recess. To measure the enzymatic activity of the gel, a small, sealed compartment was created in front of the thin gel by positioning a rubber gasket (B) within chamber (A) and pressing it in position with a second plastic chamber (C). This second chamber also had a round quartz window that was aligned with the window in the first chamber. The chamber assembly could be mounted vertically in the measuring compartment of a Cary 118C spectrophotometer. The solution bathing the polymer film within the assembled chamber could be changed rapidly and completely through the perfusion lines at the top and bottom of the chamber. FIG.
then gently lowered onto the gel drop. A thin polymer film formed in the recess between the quartz window surface and the surface of the Teflon sheath. The film gelled and 10 min later the cylindrical chamber was opened, the brass block was removed, and the polymer film was washed in place with three changes of the Hepes/EDTA solution. The plastic chamber with the thin film was then immersed in 250 ml of Hepes/EDTA solution and maintained at 4°C. The solution was changed 30 min later and the enzymatic activity of the film was then measured. To assay the enzymatic activity of the thin gels, the plastic chamber with the polymerized gel was brought to room temperature. The second Plexiglas chamber in the device and a rubber gasket were then positioned against the first chamber and clamped in
ENZYME
ELECTRODE
MEASUREMENT
place thus creating a small (about 200 gl), sealed space in front of the acrylamide film (see Fig. 1). The assembly was mounted in the measuring compartment of the spectrophotometer and maintained at room temperature. While monitoring the 340~nm absorbance of the film, the space in front of the gel was first filled with a solution of 0.2 M Hepes, 0.2 mM EDTA, and 8 mM NAD at pH 8.0. After establishing an absorbance baseline the solution in the assembly was rapidly exchanged for one of the same composition but now containing 25 mM L-glutamate.
OF
L-GLUTAMATE
191
Selection of Parameters of Acrylamide Polymerization
permeable to water and solutes as the concentrations of monomer and crosslinker increase (28,29). Given our requirement of high permeability to small substrates and moderately high mechanical rigidity, we elected to produce gels of 2% w/w crosslinker to acrylamide ratio and 8% w/v acrylamide content (T = 8, C = 2 (30)). The equivalent mean pore radius of these gels has been measured to be approximately 0.2 pm (28). Because of the need to cast electrodes of various geometries, we required the polymer-forming solution to remain fluid for a relatively long and well defined period of time. Gelling time, the time interval between the addition of the last component to the gel forming solution and the gelling of the polymer (see Materials and Methods) is determined, in part, by the concentrations of the polymerization catalyst, ammonium persulfate, and TEMED. These agents can also potentially cause loss of enzymatic activity in GDH. We investigated in parallel the effects of varying concentrations of the catalysts on both gelling time and on GDH activity in solution. Ammonium persulfate produced some enzyme damage even at the lowest concentrations tested. Therefore, we employed it at the lowest concentration sufficient to obtain gels, 0.1% w/v. The concentration of TEMED tested was in the range of 0.4 to 2.5% v/v. Over this range the amine did not produce a detectable loss of enzymatic activity, while gelling times shortened progressively as the concentration increased. For convenience we elected to produce gels with 0.8% v/v TEMED. At this concentration, the optimum gels, produced as described under Materials and Methods, had gelling times of 6 min 9 s f 43 s (mean + SD, IZ = 103). Whenever gels failed to form within this time period we discarded them.
We elected to produce gels by copolymerizing acrylamide with the crosslinker methylenebisacrylamide. In general, the gels become stronger, more rigid, and less
Covalent Immobilization of GDH. The Use of NAS Enzyme immobilization by physical entrapment in polyacrylamide gels is inade-
RESULTS
Bovine liver glutamate dehydrogenase catalyzes the oxidation of glutamate with a stoichiometric reduction of NAD into NADH (24-26). To produce an enzyme electrode it is necessary first to immobilize the enzyme in an appropriate physical support. In our application this support must meet a number of criteria: (a) it must be freely permeable to the substrates and cosubstrates of the reaction, while fully retaining the enzyme; (b) it must be fluid during some stage of its production so that by casting methods we may be able to obtain electrodes of any desired size and shape; (c) its end state must be rigid and mechanically stable; and (d) the process of enzyme immobilization should damage the protein in the least possible and should yield a stable state of the enzyme. There exists a wide choice of support materials and immobilization techniques (13,14,27). Consideration of the literature suggested that our requirements could be met best by using polyacrylamide gels as support materials and by covalently linking GDH to the gel (23).
192
KORENBROT,
PERRY, AND COPENHAGEN
quate because there is continuous protein washout from the polymer (3 1,32). This limitation can be overcome by covalently linking the protein to the acrylamide polymer through the use of N-acryloxysuccinimide (23,33). The use of NAS in the immobilization of GDH poses a problem because acylation of amine groups, precisely the chemical reaction that occurs between NAS and GDH, inhibits GDH enzymatic activity (25,34). Therefore, the gain obtained by the increased retention of GDH in the gel with the use of NAS may be negated by the loss of enzymatic activity. However, since GDH inactivation can be expected to increase proportionally with the concentration of NAS and since the efficiency of protein retention in the gel should also increase proportionally with NAS concentration, it should be possible to determine empirically an optimum concentration of NAS at which its beneficial and damaging features balance. In Fig. 2 we illustrate the results of experiments in which GDH retention and enzymatic activity were measured in gels produced in the presence of varying NAS concentrations. Protective agents are also present in the course of enzyme immobilization. The chemical identity and concentration of the protective agent is critical and its selection is detailed below. In the experiments illustrated in Fig. 2 the acylation reaction was carried out in the presence of Pipes (200 mM), EDTA (0.2 mM), and NADH (2 mM) at pH 6.5. At all concentrations of NAS tested, the reaction was carried out for 10 min prior to the addition of acrylamide. Spectrophotometric measurement of the release of N-hydrosuccinimide ions carried out as described by Adalsteinsson et al. (23) indicated that this time was sufficient to allow the acylation reaction to reach completion at all NAS concentrations tested. Inspection of Fig. 2 shows that, as expected, the efficiency of GDH retention in the gel increases with NAS concentration while its enzymatic activity decreases. Based on the experimental
-60
L5 F 5
-50
= m
-40
p
-30
I z 5
-20
o-o
2 4
6
NAS
CONCENTRATION
8 10 12 14 16 18 20 22 24 (mt.4)
FIG. 2. Effects of varying NAS concentration in the gel-forming solution on protein retention and GDH enzymatic activity of the gels. Protein retained in the gel (A) after standard washing procedures is expressed as a fraction of the amount of protein added to the gel-forming solution, 2 mg in these experiments. The specific enzymatic activity ofthe gel (0) is expressed as a fraction of that of the GDH solution used to produce the gel and assayed in parallel with the gel. The points are the average of6 to 12 individual measurements. the bars are SD.
data shown we elected to produce gels in the presence of 8 mM NAS. At this concentration the opposing effects of NAS on the properties of the enzyme gel are nearly in balance and the product of enzyme retention and specific enzymatic activity is nearly maximal.
Protection of GDH Enzymatic Activity in the Course of Gel Polymerization The enzymatic activity of GDH can be potentially inactivated in the course of acrylamide polymerization by other chemical reactions in addition to acylation by NAS. Whitesides et al. (32) have considered in detail some of the reactions likely to damage enzymes during acrylamide polymerization. As a first step to minimize enzyme deactivation we produced gels at 4°C with careful
ENZYME TABLE
ELECTRODE
MEASUREMENT
I
THE EFFECTSOF VARIOUS GOOD BUFFERSON THE GDH ACTIVITY IN GELS’ Buffer Pipes Hepes Mes Mops
Relative enzymatic activity’ 47.65 4.85 4.90 5.07
i 4.47c +- 3.11 + 1.58 * 1.19
’ All buffers were tested at a concentration of 200 mM at pH 6.5. * Relative enzymatic activity is expressed as the ratio of the specific activity measured in the gel over that measured in parallel in solution. ‘Each data point is the average of 8 measurements IL SD.
OF L-GLUTAMATE
193
The weakest buffer tested, Hepes, was not a more effective protector even when tested at 10 times higher concentration. Further, Table 2 details the effectiveness of 200 mM Pipes to protect enzymatic activity at various pH values. Although the most effective protection was obtained at pH 6.0, this pH is somewhat distant from the enzymatic pH optimum of about 7.5 (37). We elected, therefore, to carry out further investigations of enzymatic protection in the presence of 200 mM Pipes buffer at pH 6.5. NADH and MD. The liver GDH is an oxidative enzyme that utilizes NADH or NAD as a coenzyme (24,25). We, therefore, tested the effectiveness of these coenzymes as protective agents. Gels were produced in the presence of varying concentrations of these molecules. Data illustrated in Fig. 3 show that NADH is indeed an effective protector of GDH. Its effectiveness increases with concentration up to 2 mM. We were unable to explore higher concentrations because NADH interferes with gel polymerization at concentrations above 2 mM. NAD in combination with ADP can also protect the GDH activity as is illustrated in Fig. 3. The effectiveness of protection by NAD at concentrations up to 4 mM is less than that obtained with 2 mM NADH.
exclusion of oxygen. In addition, enzyme inactivation was also reduced by the use of protective agents. We investigated the effectiveness of a number of GDH cofactors and activators as protectors of the enzyme in the course of its immobilization. pH bu&ring agents. Several tertiary amines, known as Good buffers (35), were tested as potential enzyme protective agents because in addition to their effectiveness in buffering pH they quench singlet oxygen (36). Singlet oxygen is generated in the course of acrylamide polymerization and is likely to cause enzyme damage (32). We investigated GDH immobilization in the presTABLE 2 ence of Hepes (pK, 7.5) Pipes (pK, 6.8), Mes THEEFFEC~S OF pH ONTHE GDH ACTIVITYINGELS~ (4-morpholineethanesulfonic acid, pK, 6. l), and Mops (4-morpholinepropanesulfonic Relative enzymatic activityb PH acid, pK, 7.2). In Table 1 we present the rela6.0 58.70 + 7.97” tive effectiveness of the buffers as protective 6.5 52.63 + 6.95 agents, all tested at pH 6.5 and at a concen7.0 48.29 f 1.86 tration of 200 mM. At this pH all molecules 7.5 35.61 f 8.25 tested are effective hydrogen-buffering 8.0 19.50 + 2.96 agents, although their buffering power is not a Immobilization solutions contained 200 mM Pipes. identical. The high concentration tested, b Relative enzymatic activity is expressed as the ratio nonetheless, should make their difference in of the specific activity measured in the gel over that buffering power less important. Pipes was measured in parallel in solution. about ninefold more effective as a protective ’ Each data point is the average of 8- I2 measurements agent than any of the other molecules tested. k SD.
194
KORENBROT,
PERRY, AND COPENHAGEN
these results, we elected to produce the GDH polymer electrodes in the presence of 2 mM NADH alone.
50 c
I
I
NADH
GDH Kinetics in the Polyacrylamide
Gel
The activity of GDH is controlled in a complex manner by a number of effector molecules (38,39). An exhaustive investigation of the kinetic features of the immobilized enzyme was beyond the scope of this study. Nonetheless, it was important to determine the kinetic behavior of the enzyme in the gel and the effects on enzyme activity of NAD and glutamate, the coenzyme and il O 0 ADDITIVE
I
I
I
I
1
2
3
4
CONCENTRATION
(mM)
FIG. 3. Effect of varying NADH and NAD concentrations in the gel-forming solution on the GDH enzymatic activity of the gel. NADH was varied between 0.1 and 2 mM. Higher concentrations of NADH were not tested because they interfered with the process of gel polymerization. NAD was varied between 0.1 and 4 mM. In the case of NAD the solution also contained ADP at a concentration fourfold higher than that of NAD. Each point is the average of 8 to 12 measurements, except the point at 2 mM which is the average of 32 measurements. Bars show the SD.
ADP, ATP, GTP, and ZnC12. The extensive literature on GDH has demonstrated that under appropriate conditions ADP, ATP, GTP, and ZnClz can act as activators of enzymatic activity (reviewed in (24-26)). We tested the effectiveness of each of these four molecules at concentrations up to 36 mM as GDH protectors during immobilization. We found all to be less effective than 2 tIIM NADH alone (data not shown). We then investigated whether these molecules when added to 2 mM NADH increased enzyme protection. Results of these experiments are shown in Fig. 4. ADP, ATP, and GTP in concentrations up to 36 mM did not enhance enzymatic protection above that obtained with NADH alone. ZnC12 reduced the ability of NADH to protect the enzyme. Based on
ADDITIVE
CONCENTRATION
(mt.4)
FIG. 4. Effects of varying concentrations of GTP (O), ATP (0), and ZnClz (A) added to 2 rnM NADH in the gel-forming solutions on the GDH enzymatic activity of the polymer. The molecules tested were added to 2 mM NADH at concentrations that ranged from 0.1 to 2 mM. The other components of the gel-forming solution were held constant at the standard concentrations given in the text. The effects of 2 mM NADH alone are also shown in the figure (m). The effects of added ADP were also tested and they were nearly the same as those of ATP, but they are not shown in the illustration. The points are the average of 4 to 8 individual measurements, except the NADH alone which is an average of 32 measurements. The bars indicate the SD.
ENZYME
ELECTRODE
MEASUREMENT
substrate in the quantitative assay of L-glutamate. In addition to the obvious need to characterize the gel for its use as an analytical tool, kinetic studies also investigate whether the immobilization procedures alter the function of the enzyme. A Lineweaver-Burk plot of the activity of immobilized GDH as a function of NAD concentration is illustrated in Fig. 5. These data were obtained in the presence of 25 mM glutamate, a concentration that saturates enzyme velocity. As shown, the data is best fitted by two intersecting straight lines. The existence of two components in the Lineweaver-Burk demonstrated in Fig. 5 is also characteristic of the activity of the enzyme in solution and has been repeatedly reported in the literature (39,40). The two kinetic states are presumed to arise from the existence in GDH of two distinct nucleotide-binding sites, but the precise molecular mechanism that gives rise to the observed change in kinetics is not understood (40-42). By fitting straight lines to the experimental data, two kinetic domains can be defined one of Kmapp
10
195
OF L-GLUTAMATE
r
01;
’
1
’
2
(L-GLUTAMATE
’
3
’
4
’
5
’
6
CONCENTRATION)-’
I 10 (NAD
I 20 CONCENTRATION)-’
I 30
I 40
I 50
hM-1)
FIG. 5. Lineweaver-Burk plot of the velocity of GDH activity in polymer gels as a function of NAD concentration. The gels were assayed in the presence of 25 mM x&8amate. Just as in solution, two kinetic components are apparent in the plot. They are presumed to arise from the existence of two distinct nucleotide-binding sites in the enzyme (42,43).
’
8
’
9
’
10
(md)
FIG. 6. Lineweaver-Burk plot of the velocity of GDH activity in the polymer gels as a function of L-glutamate concentration. The gels were assayed in the presence of 8 mM NAD.
= 1.14 mM and V,,, = 1.455 pmol/min . mg protein and the other of KmaPP = 0.12 mM and Max = 0.34 pmol/min . mg protein. GDH activity in the gel as a function of L-glutamate concentration assayed in the standard conditions and in the presence of 8 mM NAD is illustrated in a LineweaverBurk plot in Fig. 6. The data were fitted with a single line from which a Kmapp = 0.85 mM * mg protein were and V,,, = 0.98 pmol/min calculated. The kinetic behavior of the enzyme immobilized in the gel, within the few kinetic parameters tested, is not different from that of the enzyme in solution. Immobilization as described here apparently does not alter significantly the function of the active, bound enzyme. Speed of Response of the Polymer
0
’
7
Gel
To measure the response time of the GDH-containing gel to a step change in glutamate concentration we constructed a small chamber that permitted the continuous monitoring of the optical absorbance of a thin, about 70 pm, gel film. A typical continuous time record of the change in absorbance at 340 nm of the gel film following a sudden change in glutamate concentration from 0 to 25 mM is shown in Fig. 7. In the record, the sudden and steady increase in absorbance, indicative of the production of NADH, is nearly simultaneous with the change in glu-
196
KORENBROT,
+Glu
(25
PERRY, AND COPENHAGEN
mM)
o-4 Time
(se4
FIG. 7. Speed of response of thin enzyme gels. The absorbance at 340 nm of the gel produced in the chamber illustrated in Fig. 1 was monitored continuously. The gel was initially bathed with the standard assay solution described in the text. That solution was rapidly exchanged for one containing 25 mM L-glutamate and 8 mM NAD. Within the resolution of the instrumentation, the absorbance of the film changed simultaneously with the change in solution.
tamate concentration and exhibits no detectable latency. The initial rate of change in absorbance is, of course, proportional to glutamate concentration for nonsaturating concentrations. The data indicate that the response of the electrode is indeed rapid, well under 500 ms, and in the experiments presented here the measured rate of response is most likely limited by the frequency bandpass of the recording instrument.
limited by the amount of enzyme present in the gel, since NAD and glutamate are in vast molar excess relative to the enzyme. To improve the analytical value of the gel we investigated the effects of increasing its enzyme content. The activity of gels as a function of their enzyme content is illustrated in Fig. 8. We tested gels that varied in protein content between 0.08 and 8 mg/ml. Enzymatic activity of the gel indeed increases proportionately with its protein content over the range tested, although the relationship is linear only at the lower concentrations of less than about 1 mg/ml. The observed nonlinear dependence of enzyme activity on enzyme concentration is unexpected for enzymes in solution, but is characteristic of immobilized enzymes and is, indeed, the kinetic advantage of immobilized over free enzymes in analytical applications ( 13). The gel specificity, that is, its ability to signal the presence of the substrate of interest and not of other structurally analogous sub-
‘r
Detection, Sensitivity, SpeciJicity,and Stability of the Polymer Gel The detection sensitivity of an enzyme electrode is ultimately limited by the kinetic characteristics of the enzyme it contains. The lowest concentration of glutamate detectable by our gels can be determined, in principle, from the glutamate KmaPP of GDH in the gel by well established theoretical methods ( 13). Calculations show that our electrode should at least detect about 3 PM glutamate. The experimental limit of sensitivity, however, is likely to be given by the signal/noise features of the instrumentation used to measure NADH absorbance and/or fluorescence. In our gels the magnitude of the optical signal is
GDH
CONCENTRATION
(mglgm
gel)
RG. 8. The effect of varying GDH concentration in the gel on its enzymatic activity. GDH concentration was varied between 0.08 and 8 mg per gram of gel by varying its concentration in the gel-forming solution. The other components of the gel-forming solution were held constant at the standard concentrations given in the text. The data points are the average of 4 to 8 measurements, bars are SD. The line was drawn by eye, except at the low concentrations where a straight line was fitted to the points. The nonlinear dependence of enzymatic activity on protein concentration at high concentrations is not observed in solution, but is characteristic of immobilized enzymes.
ENZYME
ELECTRODE
MEASUREMENT
strates, should be entirely defined by the specificity of the enzyme. GDH is highly selective in its deamination reaction for L-glutamate over other substrates (reviewed in (26)). For neurotransmitter studies it is important to establish the selectivity of the GDH in the gel for L-glutamate over structurally similar putative neurotransmitters. Under our standard assay conditions, there was no spectrophotometrically detectable signal in the gel (less than 0.0005 OD/min) in response to the application of up to 435 mM D-ghtamate, 435 mM L-aspartate, 350 mM D-aspartate, and 400 mM N-acetyl-Lhistidine. Since L-glutamate in concentrations of 5 pM produces an easily detectable signal, the response for L-glutamate of the gel exceeds that for D-glutamate, D- and Laspartate, and N-acetyl-L-histidine by at least 1OOO:l. To determine the long-term stability of the electrodes, we produced gels, collected and washed them as usual, and then resuspended them in the normal assay solution containing 8 mM NAD and no glutamate. This suspension was stored at 4°C. The presence of NAD in the storage medium was necessary to maintain activity in the gel over a long term of time. Because NAD is not stable in solution for long, we refreshed the NAD in the gel suspension every 3 to 4 days. At daily intervals aliquots were removed from the gel suspension, exhaustively washed free of NADH, and assayed for their enzymatic activity in the usual manner. We found the gels to be stable, that is, not to lose any appreciable enzymatic activity, for up to 25 days after their production. We did not explore longer time periods. Also, we did not explore the behavior of the gel on repeated use. DISCUSSION
With detailed attention to methodological procedures we have produced hydrophilic and permeant acrylamide polymers that contain covalently immobilized bovine liver
OF
L-GLUTAMATE
197
GDH with preservation of about 50% of the activity of the enzyme. These polymers are the basis of enzyme electrodes to measure L-glutamate that are sensitive, specific, rapid, and stable. The immobilization of GDH described here represents a specific application of a methodological concept pioneered by Adalsteinsson et al. (23). The method developed here can be generalized to the covalent immobilization of other enzymes to produce electrodes for other substrates. To preserve enzymatic activity in the gels several general procedural conditions should be met. First, oxygen should be carefully excluded from the reaction mixtures and all reactions should be carried out at 4°C. Second, the reaction of enzyme with the covalent immobilizing agent, NAS, should be investigated over a range of concentrations to optimize the efficiency of retention while minimizing the potential inactivation of the enzyme. Third, the acrylamide monomers and the catalytic agents in the free radical polymerization of acrylamide should be used at the lowest acceptable concentration consistent with the desired mechanical strength, permeability, and gelling time for the polymer. Fourth, the enzyme should be protected from inactivation by substrates, cofactors, and coenzymes. To measure the concentration of L-glutamate with the GDH enzyme gels there are two reaction products that can be measured, NADH and NH4. The first can be monitored optically by its absorbance or its fluorescence, while the second can be monitored with an ion-specific electrode (13). In the present study we elected to follow photometrically the production of NADH by measuring the absorbance of the gel at 340 nm. Optical methods have a disadvantage when compared to electrochemical ones in that the necessary instrumentation is often more complex. This experimental option is particularly important when considering the application of these enzyme gels to studies of neurotransmitter release in single cells. We wish
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to point out that optical methods offer a unique advantage over electrochemical ones that justifies the necessary instrumental complexity: The delays imposed by the diffusion of reaction products from the reaction site to the electrode surface are eliminated (13). The response time of the electrode is, therefore, likely to be limited only by the diffusion rate of the substrate through the gel. Indeed, we have shown here the experimental response time of our gels to be rapid, under 500 ms, and likely to be limited by the instrumental frequency response characteristics of the spectrophotometer used. GDH has been used in glutamate-detecting polymers in several previous designs (17-22) and has also been used in solution in assays of L-glutamate (15). The principal advantage of the design described here is the ability to cast electrodes of any desired shape because the gel making solution is fluid for a relatively long and reproducible amount of time. Two previous reports have investigated in detail the kinetic consequences of immobilizing GDH. Barbotin and Breuil (43) immobilized GDH in an albumin film using glutaraldehyde as crosslinker, whereas Julliard et al. (44) bound GDH to a collagen film. The authors found some modification of the control features of the enzyme upon immobilization. The qualitative and quantitative nature of the changes, however, were different in each of the two methods. The allosteric and regulatory features of GDH were not exhaustively examined in the polyacrylamide gels described here. To the extent that they were investigated, the features of the enzyme in the gel were indistinguishable from those in solution. More extensive studies might possibly reveal some kinetic changes. However, for the purpose of analytical investigation the gels described here provide a good basis for reliable and stable enzyme electrodes to be used in neurochemical studies. ACKNOWLEDGMENTS This study was supported by Grants EY01586 (J.I.K.) and EY01869 (D.R.C.) from the NIH and a John S.
Adams award to D.R.C. from Research to Prevent Blindness.
REFERENCES 1. Gaddum, J. H. (196 I) J. Physiol. 155, 1-2. Philippu, A. (1984) in Measurements of Neurotransmitter Release in Vivo (C. A. Marsden, Ed.), pp. 3-38, Wiley, New York. 3. Delgado, J. M. R., DeFeudis, F. V., Roth, R. H., Ryugo, D. K., and Mitruka, B. M. (1972) Arch. Int. Pharmacodyn. Ther. 198, 9-2 1. 4. Ungerstedt, U. (1984) in Measurements of Neurotransmitter Release in Vivo (C. A. Marsden, Ed.), pp. 8 I-104, Wiley, New York. 5. Mitchell, J. F. (1963) J. Physiol. 165, 98-l 16. 6. Marsden, C. A., Brazell, M. P., and Maidment, N. T. (1984) in Measurements of Neurotransmitter Release in Vivo (C. A. Marsden, Ed.), pp. 127- 149, Wiley, New York. 7. Schenk, J. O., Miller, E., Rice, M. E., and Adams, R. N. (1983) Brain Rex 277, l-8. 8. Young, S. H., and Poo. M.-M. (1983) Nature (Lon2.
don) 305634-637.
9. Hume, R. I., Role, L. W., and Fishbach, G. D. (1983) Nature (London) 305632-634. 10. Miller, D. L., and Korenbrot, J. I. (1984) Abstr. Biophys. Sot. Meet.; Biophys. J. 45, 34 1a. 11. Korenbrot, J. I., and Miller, D. L. (1986) Neurosci. Res. 4, Sl l-S34. 12. Miller, A. M., and Schwartz, E. A. (1983) J. Physiol. 334,325-349.
13. Carr, P. W., and Bowers, L. D. (1980) Immobilized Enzymes in Analytical and Clinical Chemistry, Wiley, New York. 14. Wingard, L. M., Katchalski-Katzir, E., and Goldstein, L. (1981) Analytical Applications of Immobilized Enzymes and Cells, Pergamon Press, New York. 15. Guilbault, G. G. (1976) Handbook of Enzymatic Methods of Analysis, Dekker, New York. 16. Clark, L. C., and Lyons, C. ( 1962) Ann. N. Y. Acad. Sci. 102, 29-45.
17. Davies. P., and Mosbach, K. (1974) Biochim. Biophys.
Acta 370, 329-338.
18. Chan, W. W. C., Davies, P., and Mosbach, K. (1976) in Ions and Enzyme Electrodes (M. Kessler, Ed.), pp. 182-207, Univ. Park Press, Baltimore, MD. 19. Guilbault, G. G., and Hrabankova, E. (1970) Anal. Chem. 42, 1779-1783. 20. Nilsson, H., Akerlund, A., and Mosbach, K. (1973) Biochim.
Biophys.
Acta. 320, 529-534.
21. Ahn, B. K., Wolfson, S. K., and Yao, S. J. (1975) Bioelectrochem Bioenergy 2, 142- 147.
ENZYME
ELECTRODE
MEASUREMENT
22. Nanjo, M., and Guilbault, G. G. (1974) Anal. Chim. Acta. 13, 367-373. 23. Adalsteinsson, O., Lamotte, A., Baddour, R. F., Colton, C. K., Pollak, A., and Whiteside, G. M. ( 19793 .I Mol. Catal. 6, I99-225. 24. Smith, E. L., Austen, B. M., Blumenthal, K. M., and Nyc, J. F. ( 1975) in The Enzymes (Boyer, P., Ed.), Vol. 11, pp. 294-366, Academic Press, New York. 25. Eisenberg, H., Josephs, R., and Reisler. E. (1976) Adv. Prot. Chem. 30, 101-181. 26. Gore, M. G. (1981) J. B&hem. 13,879-886. 27. Mosbach, K. (1976) in Methods in Enzymology (Mosbach, K., Ed.), Vol. 44, Academic Press, New York. 28. White, M. L.(l960) J. Phys. Chem. 64, 1563-1567. 29. Fawcett, J. S., and Morris, C. J. 0. R. (1966) Sep. Sci. 1, 9-26. 30. Rodbard, D., and Crambach, A. (197 1) Anal. Biothem. 40,95-134. 31. Frieden, C. (1959) J. Biol. Chem. 234,815-820. 32. Whitesides, G. M.. Lamotte, A. L., Adalsteinsson, 0.. Baddour, R. F., Chmurny, A. C., Colton, C. K., and Pollack, A. (1979) J. Mol. Cafal. 6, 177-198.
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33. Schnaar, R. L., and Lee, Y. C. (1975) Biochemistry 14,1535-1544.
34. Markau, K., Schneider, J., and Sund, H. (1972) FEBS Lett. 24, 32-36. 35. Good, N. E., Winget, G. D., Winter, W., Conally, T. N., Izawa, S., and Sir& R. M. M. (1966) Biochemistry 5,467-477. 36. Ogryslo, E. A., and Tang, C. W. (1970) J. Amer. Chem. Sot. 92,5034-5038. 37. DiPrisco, R. (1975) Arch. Biochem. Bioph.vs. 171, 604-6 38.
39. 40. 41. 42. 43. 44.
12.
Markau, K., Schneider, J., and Sund, H. (1972) FEBS Left. 24,32-36. Bailey. J., Bell, E. T., and Bell, J. E. (1982) J. Biol. Chem. 257, 5579-5583. Frieden, C. (1959) J. Biol. Chem. 234,815-820. Jallon, J. M., and Iwatsubo, M. (1971) Biochem. Biophys. Res. Commun. 45,964-97 1. Sund, H., Dieter, H., Koberstein, R., and Rasched, I. (1977) J. Mol. Cutal. 2, l-23. Barbotin, J. N., and Breuil, M. (1978) Biochim. Biophys. Acta 525, 18-27. Julliard, J. H., Godinot, C., and Gautheron, D. C. (1971) FEBS Left. 14, 185-188.
BIOCHEMISTRY
Development
161, 187-199 (1987)
and Characterization of a Polymer Gel with an Immobilized Enzyme to Measure L-Glutamate
JUANI.KORENBROT,RUTHPERRY,ANDDAVID Departments
of Physiology,
Biochemistry, and Ophthalmology, San Francisco, California
R.~OPENHAGEN University 94143
of California
Medical
School,
Received March 25, 1986 Glutamate dehydrogenase (GDH) is used in an enzyme electrode to measure L-glutamate. GDH is covalently immobilized in a hydrophilic, permeable, and semirigid gel produced by the copolymerization of polyacrylamide and N-acryloxysuccinimide. Experimental conditions necessary to retain GDH in the gel with high efficiency and minimum denaturation are optimized. The abilities of enzymatic cofactors and coenzymes, NADH, NAD, ATP. ADP, GTP, and ZnC&, to protect the enzyme during immobilization are explored. Under optimum experimental procedures an enzyme-containing gel is produced that is reproducible and long lasting in its functional behavior. The gel responds to the presence of L-glutamate with high velocity, the delay being less than 500 ms; high specificity, being lOOO-fold more responsive to L-glutamate than D-ghtaIIIate, D or L-aspartate, and N-acetylhistidine; and high sensitivity, a concentration of about 3 PM can be measured. 0 1987 Academic press, IX. KEY WORDS: immobilized enzyme; enzyme electrode; L-glutamate; glutamate dehydrogenase; neurochemistry; amino acid analysis.
The measurement of the release of neurotransmitter molecules by neurons is an experimental technique that allows the determination of the chemical identity of these molecules and the study of the physiological control of their cellular release. For these measurements, the medium bathing the neuron is subjected to quantitative chemical analysis. Typically, either of two experimental strategies are used. In one method, a relatively large volume of extracellular medium is collected over a period of time and then chemically analyzed. The extracellular medium can be collected in vivo, for example with push-pull cannulaes (1,2), by local dialysis (3,4), or with collecting cups (5), or in vitro, for example by recovering the medium in which neurons are maintained in culture. The second strategy depends on the continuous sampling of the medium in the extracellular space with an appropriate probe placed near the neuron. In in vivo experiments, for
example, polarographic electrodes are placed within the nervous tissue (6,7). In in vitro experiments “bioelectrodes,” constructed by sealing the opening of a “patch” micropipet with a cell membrane fragment responsive to the selected neurotransmitter, are placed near the surface of the neuron (8,9). The continuous chemical sampling of the extracellular medium is the preferred method because it permits the resolution of the kinetics of neurotransmitter release. This experimental advantage, however, can be realized only if the analytical probe in the extracellular space is sufficiently small, specific, and sensitive. These problems can, in principle, be resolved by appropriate design of the probe. The problem of low sensitivity can be overcome by using probes that respond to concentration changes in a design that minimizes extracellular volume so that very small mole changes produce large concentration changes. We have used this strategy, for ex-
187
0003-2697187 $3.00 Copyright0 1987 by Academic All rights of reproduction
Press, Inc. in any form reserved.
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KORENBROT,
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ample, to measure the small, light-dependent release of Ca ions by single vertebrate rod photoreceptor cells (IO, 11). Because of our interest in rod photoreceptors, a neuron believed to use L-glutamate as a neurotransmitter ( 12), we undertook the development of an experimental system to measure L-glutamate with a concentration-sensitive probe. We elected to develop an enzyme electrode for L-glutamate. Enzyme electrodes are a general class of analytical probes that respond to concentration changes and can be made to be highly sensitive, specific, and small (13-15). In an enzyme electrode, a selected enzyme is physically immobilized in a support placed on or near a physical detector. The molecule to be measured is acted upon by the immobilized enzyme and in this reaction products or cosubstrates, that are readily measured by the physical probe, are produced or consumed in stoichiometric amounts (16). L-Glutamate-sensitive enzyme electrodes based on the activity of either glutamate dehydrogenase ( 17,18), glutamate decarboxylase ( 19-2 1), or glutamate oxidase (22) have been reported previously. Previous available designs, however, proved incompatible with our needs, in particular because we required the ability to produce a microprobe having a diameter less than 50 pm. We describe here an electrode based on the covalent immobilization of glutamate dehydrogenase in a polyacrylamide polymer. We propose that such an electrode, when applied under conditions that minimize extracellular volume ( 10,l l), will allow the study of glutamate release by intact single neurons. This method, therefore, should be of use to those interested in the role of glutamate, a neurotransmitter found throughout the nervous system. More generally, we propose that the rationale of the method should make it applicable to the study of other biologically active molecules secreted by neurons or other cell types for which an enzyme electrode can be designed.
MATERIALS
Materials
AND
METHODS
and Apparatus
Glutamate dehydrogenase (GDH)’ (EC 1.4.1.3) purified from beef liver was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). N-Acryloxysuccinimide (NAS) was synthesized following the method of Adalsteinsson et al. (23). Acrylamide (99.9%), N,N’-methylenebisacrylamide (Bis), ZV,N,N’,N’-tetramethylethylenediamine (TEMED), and ammonium persulfate were obtained from Bio-Rad (Richmond, CA). Pipes (piperazine-N,N’bis(2-ethanesulfonic acid)) was from P-L Biochemicals (Milwaukee, WI), and all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Spectrophotometric studies were conducted in a Cary 118C instrument (Varian Instruments, Mountain View, CA). Methods Covalent immobilization of GDH in an acrylamide polymer. The following seven stock solutions were prepared: S 1, 0.65 M Pipes and 0.65 mM EDTA, adjusted to pH 6.5 with NaOH. S2, acrylamide (295 mg/ml) and Bis (6.15 mg/ml) in distilled water. S3, 20 mM NADH in distilled water. S4, TEMED (20% v/v) in distilled water. S5, potassium persulfate in distilled water (25 mg/ml). S6, GDH (10 mg/ml) in 30 mM potassium phosphate buffer at pH 7.0. S7, 20 mM NAS in (CH&SO (dimethyl sulfoxide). Stocks 3-6 were prepared fresh each time. All solutions, except S7, were maintained at 4°C. The solutions listed above are those ’ Abbreviations used: GDH, glutamate dehydrogenase; NAS, N-acryloxysuccinimide; Bis, N,N’methylenebisacrylamide; TEMED, N,N,N’,N’tetramethylenediamine; Pipes, piperazine-N,N’-bis(2ethanesulfonic acid); Hepes, 4-(2-hydroxyethyl)I-piperazineethanesulfonic acid; Mes, 4-morpholineethanesulfonic acid: Mops, 4-morpholineethanesulfonic acid.
ENZYME
ELECTRODE
MEASUREMENT
used to produce optimum gels. Enzyme cofactors and activators tested were added in stock S3. Gel production was carried out in a cold room at 4°C. Because the polymerization of acrylamide is inhibited by molecular oxygen we degassed all stock solutions and then saturated them with nitrogen immediately before use. Mixing of stock solutions and gel polymerization were conducted under a nitrogen atmosphere within an enclosed cylindrical Plexiglas chamber ( 14 cm in diameter and 10 cm in height). Degassed solutions Sl-S6 were aliquoted into each of six vials through a small opening in the plastic chamber. Solutions were then transferred from each vial into a seventh, stirred vial in the following volume and sequence: 0.320 ml of S1,0.2 ml of S6, 0.1 ml of S3, and 0.04 ml of S7. Exactly 10 min later 0.273 ml of S2,0.04 ml of S4, and 0.04 ml of S5 were added. When we tested the effects of various chemical constituents and their concentrations on gel production, the stock solutions were adjusted such that the sequence and volume of their addition was unchanged.
Gelfing time and recovery of the polymer. Gelling time was defined as the time interval between the addition of solution S5 and the moment at which the rotation of the stirring bar in the mixing vial ceased. For any given set of experimental conditions this time was reproducible to within + 15%. If gels failed to form within this expected time interval they were discarded. The rare failures could usually be traced to either inadequate removal of oxygen or contamination of stock solutions. Ten minutes after its polymerization the gel was covered in the mixing vial with 0.5 ml of a solution of 0.2 M Hepes (4-(2-hydroxyethyl)1 -piperazineethanesulfonic acid) and 0.2 mM EDTA. Two minutes later the Plexiglas chamber was opened and the gel was transferred with the aid of 25 ml of the Hepes/EDTA solution to the glass vessel of an homogenizer.
OF
L-GLUTAMATE
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Gel washing and determination of e@ciency of protein retention. The enzyme-containing polymer was thoroughly homogenized at 4°C with a motor-driven, tight-fitting Teflon pestle (A. H. Thomas Co. Philadelphia, PA). The homogenate was mixed with 25 ml of the Hepes/EDTA solution and centrifuged at 4°C for 55 min at 10,OOOg. The supernatant was collected and the gel pellet was washed twice again by resuspension and centrifugation with Hepes/ EDTA solution. The protein content of each of these supernatants was measured colorimetrically using GDH as the standard (Bradford protein assay, Bio-Rad Labs, Richmond CA). The protein content of the gel was determined by subtracting the sum of the protein amounts measured in all supernatants from the protein amount added to the gelforming solution. Efficiency of protein retention in the gel was defined as the ratio of the amount of protein retained by the gel to that added to the gel-forming solution.
Assay of enzymatic activity in the gel. GDH activity was measured spectrophotometrically at room temperature by determining the initial rate of reduction of NAD into NADH. All assays, except those in the stability studies, were completed within 24 h of the production of the gel. Following the last wash and centrifugation, the gel pellet was carefully weighed and resuspended in 0.2 M Hepes, 0.2 mM EDTA, and NAD, pH 8.0, at 0.1 to 0.2 mg gel/ml. The concentration of NAD in this solution was varied in those experiments in which we investigated its effects on enzyme kinetics, otherwise it was 8 mM. The gel suspension was maintained at 4°C until shortly before it was assayed. To assay GDH activity, the gel suspension was vortexed and a 0.5-ml aliquot was rapidly mixed in a quartz cuvette with an equal volume of the Hepes/EDTA/NAD solution containing L-glutamate. The final concentration of L-glutamate in this solution was varied in those experiments in which we investigated its effects on enzyme kinetics,
190
KORENBROT,
PERRY, AND COPENHAGEN
otherwise it was 25 mM. The quartz cuvette was immediately placed in the measuring compartment of a spectrophotometer and its absorbance at 340 nm was measured as a function of time. Enzymatic activity was calculated from the absorbance data given a molar extinction coefficient of NADH at 340 nm of 6.22 X lo6 It cm/M. The specific enzymatic activity in repeat assays of the same gel varied within about 5%; however, this value varied by as much as 20% among different gels produced by the same protocol. In order to average and compare gels produced under varying experimental conditions on different dates we used the following quantitative protocol. The enzyme solution used in the production of each gel (S2 above) was assayed in parallel with the gels and its specific enzymatic activity was thus determined. To normalize and compare data we expressed the specific activity in the gel as a fraction of the specific activity of the enzyme in the solution used to produce that gel. Speed of response of thin gels. To determine the speed of response of the gel to step changes in glutamate concentration we assayed the enzymatic activity of thin, 70 pm, polymer films produced in the device illustrated in Fig. 1. This device could be placed in the measuring compartment of the spectrophotometer and the glutamate concentration in the medium bathing the gel within the device could be changed rapidly. The device consisted of two Plexiglas chambers. In one of them, a round quartz window was mounted with a 70-pm recess relative to the wall of the chamber. A thin film of enzymecontaining polymer was cast in that recess. To do so, this chamber was placed horizontally within the nitrogen-saturated cylindrical chamber described above. The gel-forming solution stocks were mixed as described and 4 min after adding S5, 200 ~1 of the still ungelled solution were placed on top of the quartz window. A heavy block of brass covered with a 3-mm-thick sheath of Teflon was
Inflow
1. Schematic drawing of the chamber used to produce thin GDH-polyacrylamide gels and to measure their enzymatic activity. One Plexiglas chamber (A) had a quartz round window(q) recessed 70 pm relative to the wall of the chamber. The polymer gel was formed within that recess. To measure the enzymatic activity of the gel, a small, sealed compartment was created in front of the thin gel by positioning a rubber gasket (B) within chamber (A) and pressing it in position with a second plastic chamber (C). This second chamber also had a round quartz window that was aligned with the window in the first chamber. The chamber assembly could be mounted vertically in the measuring compartment of a Cary 118C spectrophotometer. The solution bathing the polymer film within the assembled chamber could be changed rapidly and completely through the perfusion lines at the top and bottom of the chamber. FIG.
then gently lowered onto the gel drop. A thin polymer film formed in the recess between the quartz window surface and the surface of the Teflon sheath. The film gelled and 10 min later the cylindrical chamber was opened, the brass block was removed, and the polymer film was washed in place with three changes of the Hepes/EDTA solution. The plastic chamber with the thin film was then immersed in 250 ml of Hepes/EDTA solution and maintained at 4°C. The solution was changed 30 min later and the enzymatic activity of the film was then measured. To assay the enzymatic activity of the thin gels, the plastic chamber with the polymerized gel was brought to room temperature. The second Plexiglas chamber in the device and a rubber gasket were then positioned against the first chamber and clamped in
ENZYME
ELECTRODE
MEASUREMENT
place thus creating a small (about 200 gl), sealed space in front of the acrylamide film (see Fig. 1). The assembly was mounted in the measuring compartment of the spectrophotometer and maintained at room temperature. While monitoring the 340~nm absorbance of the film, the space in front of the gel was first filled with a solution of 0.2 M Hepes, 0.2 mM EDTA, and 8 mM NAD at pH 8.0. After establishing an absorbance baseline the solution in the assembly was rapidly exchanged for one of the same composition but now containing 25 mM L-glutamate.
OF
L-GLUTAMATE
191
Selection of Parameters of Acrylamide Polymerization
permeable to water and solutes as the concentrations of monomer and crosslinker increase (28,29). Given our requirement of high permeability to small substrates and moderately high mechanical rigidity, we elected to produce gels of 2% w/w crosslinker to acrylamide ratio and 8% w/v acrylamide content (T = 8, C = 2 (30)). The equivalent mean pore radius of these gels has been measured to be approximately 0.2 pm (28). Because of the need to cast electrodes of various geometries, we required the polymer-forming solution to remain fluid for a relatively long and well defined period of time. Gelling time, the time interval between the addition of the last component to the gel forming solution and the gelling of the polymer (see Materials and Methods) is determined, in part, by the concentrations of the polymerization catalyst, ammonium persulfate, and TEMED. These agents can also potentially cause loss of enzymatic activity in GDH. We investigated in parallel the effects of varying concentrations of the catalysts on both gelling time and on GDH activity in solution. Ammonium persulfate produced some enzyme damage even at the lowest concentrations tested. Therefore, we employed it at the lowest concentration sufficient to obtain gels, 0.1% w/v. The concentration of TEMED tested was in the range of 0.4 to 2.5% v/v. Over this range the amine did not produce a detectable loss of enzymatic activity, while gelling times shortened progressively as the concentration increased. For convenience we elected to produce gels with 0.8% v/v TEMED. At this concentration, the optimum gels, produced as described under Materials and Methods, had gelling times of 6 min 9 s f 43 s (mean + SD, IZ = 103). Whenever gels failed to form within this time period we discarded them.
We elected to produce gels by copolymerizing acrylamide with the crosslinker methylenebisacrylamide. In general, the gels become stronger, more rigid, and less
Covalent Immobilization of GDH. The Use of NAS Enzyme immobilization by physical entrapment in polyacrylamide gels is inade-
RESULTS
Bovine liver glutamate dehydrogenase catalyzes the oxidation of glutamate with a stoichiometric reduction of NAD into NADH (24-26). To produce an enzyme electrode it is necessary first to immobilize the enzyme in an appropriate physical support. In our application this support must meet a number of criteria: (a) it must be freely permeable to the substrates and cosubstrates of the reaction, while fully retaining the enzyme; (b) it must be fluid during some stage of its production so that by casting methods we may be able to obtain electrodes of any desired size and shape; (c) its end state must be rigid and mechanically stable; and (d) the process of enzyme immobilization should damage the protein in the least possible and should yield a stable state of the enzyme. There exists a wide choice of support materials and immobilization techniques (13,14,27). Consideration of the literature suggested that our requirements could be met best by using polyacrylamide gels as support materials and by covalently linking GDH to the gel (23).
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KORENBROT,
PERRY, AND COPENHAGEN
quate because there is continuous protein washout from the polymer (3 1,32). This limitation can be overcome by covalently linking the protein to the acrylamide polymer through the use of N-acryloxysuccinimide (23,33). The use of NAS in the immobilization of GDH poses a problem because acylation of amine groups, precisely the chemical reaction that occurs between NAS and GDH, inhibits GDH enzymatic activity (25,34). Therefore, the gain obtained by the increased retention of GDH in the gel with the use of NAS may be negated by the loss of enzymatic activity. However, since GDH inactivation can be expected to increase proportionally with the concentration of NAS and since the efficiency of protein retention in the gel should also increase proportionally with NAS concentration, it should be possible to determine empirically an optimum concentration of NAS at which its beneficial and damaging features balance. In Fig. 2 we illustrate the results of experiments in which GDH retention and enzymatic activity were measured in gels produced in the presence of varying NAS concentrations. Protective agents are also present in the course of enzyme immobilization. The chemical identity and concentration of the protective agent is critical and its selection is detailed below. In the experiments illustrated in Fig. 2 the acylation reaction was carried out in the presence of Pipes (200 mM), EDTA (0.2 mM), and NADH (2 mM) at pH 6.5. At all concentrations of NAS tested, the reaction was carried out for 10 min prior to the addition of acrylamide. Spectrophotometric measurement of the release of N-hydrosuccinimide ions carried out as described by Adalsteinsson et al. (23) indicated that this time was sufficient to allow the acylation reaction to reach completion at all NAS concentrations tested. Inspection of Fig. 2 shows that, as expected, the efficiency of GDH retention in the gel increases with NAS concentration while its enzymatic activity decreases. Based on the experimental
-60
L5 F 5
-50
= m
-40
p
-30
I z 5
-20
o-o
2 4
6
NAS
CONCENTRATION
8 10 12 14 16 18 20 22 24 (mt.4)
FIG. 2. Effects of varying NAS concentration in the gel-forming solution on protein retention and GDH enzymatic activity of the gels. Protein retained in the gel (A) after standard washing procedures is expressed as a fraction of the amount of protein added to the gel-forming solution, 2 mg in these experiments. The specific enzymatic activity ofthe gel (0) is expressed as a fraction of that of the GDH solution used to produce the gel and assayed in parallel with the gel. The points are the average of6 to 12 individual measurements. the bars are SD.
data shown we elected to produce gels in the presence of 8 mM NAS. At this concentration the opposing effects of NAS on the properties of the enzyme gel are nearly in balance and the product of enzyme retention and specific enzymatic activity is nearly maximal.
Protection of GDH Enzymatic Activity in the Course of Gel Polymerization The enzymatic activity of GDH can be potentially inactivated in the course of acrylamide polymerization by other chemical reactions in addition to acylation by NAS. Whitesides et al. (32) have considered in detail some of the reactions likely to damage enzymes during acrylamide polymerization. As a first step to minimize enzyme deactivation we produced gels at 4°C with careful
ENZYME TABLE
ELECTRODE
MEASUREMENT
I
THE EFFECTSOF VARIOUS GOOD BUFFERSON THE GDH ACTIVITY IN GELS’ Buffer Pipes Hepes Mes Mops
Relative enzymatic activity’ 47.65 4.85 4.90 5.07
i 4.47c +- 3.11 + 1.58 * 1.19
’ All buffers were tested at a concentration of 200 mM at pH 6.5. * Relative enzymatic activity is expressed as the ratio of the specific activity measured in the gel over that measured in parallel in solution. ‘Each data point is the average of 8 measurements IL SD.
OF L-GLUTAMATE
193
The weakest buffer tested, Hepes, was not a more effective protector even when tested at 10 times higher concentration. Further, Table 2 details the effectiveness of 200 mM Pipes to protect enzymatic activity at various pH values. Although the most effective protection was obtained at pH 6.0, this pH is somewhat distant from the enzymatic pH optimum of about 7.5 (37). We elected, therefore, to carry out further investigations of enzymatic protection in the presence of 200 mM Pipes buffer at pH 6.5. NADH and MD. The liver GDH is an oxidative enzyme that utilizes NADH or NAD as a coenzyme (24,25). We, therefore, tested the effectiveness of these coenzymes as protective agents. Gels were produced in the presence of varying concentrations of these molecules. Data illustrated in Fig. 3 show that NADH is indeed an effective protector of GDH. Its effectiveness increases with concentration up to 2 mM. We were unable to explore higher concentrations because NADH interferes with gel polymerization at concentrations above 2 mM. NAD in combination with ADP can also protect the GDH activity as is illustrated in Fig. 3. The effectiveness of protection by NAD at concentrations up to 4 mM is less than that obtained with 2 mM NADH.
exclusion of oxygen. In addition, enzyme inactivation was also reduced by the use of protective agents. We investigated the effectiveness of a number of GDH cofactors and activators as protectors of the enzyme in the course of its immobilization. pH bu&ring agents. Several tertiary amines, known as Good buffers (35), were tested as potential enzyme protective agents because in addition to their effectiveness in buffering pH they quench singlet oxygen (36). Singlet oxygen is generated in the course of acrylamide polymerization and is likely to cause enzyme damage (32). We investigated GDH immobilization in the presTABLE 2 ence of Hepes (pK, 7.5) Pipes (pK, 6.8), Mes THEEFFEC~S OF pH ONTHE GDH ACTIVITYINGELS~ (4-morpholineethanesulfonic acid, pK, 6. l), and Mops (4-morpholinepropanesulfonic Relative enzymatic activityb PH acid, pK, 7.2). In Table 1 we present the rela6.0 58.70 + 7.97” tive effectiveness of the buffers as protective 6.5 52.63 + 6.95 agents, all tested at pH 6.5 and at a concen7.0 48.29 f 1.86 tration of 200 mM. At this pH all molecules 7.5 35.61 f 8.25 tested are effective hydrogen-buffering 8.0 19.50 + 2.96 agents, although their buffering power is not a Immobilization solutions contained 200 mM Pipes. identical. The high concentration tested, b Relative enzymatic activity is expressed as the ratio nonetheless, should make their difference in of the specific activity measured in the gel over that buffering power less important. Pipes was measured in parallel in solution. about ninefold more effective as a protective ’ Each data point is the average of 8- I2 measurements agent than any of the other molecules tested. k SD.
194
KORENBROT,
PERRY, AND COPENHAGEN
these results, we elected to produce the GDH polymer electrodes in the presence of 2 mM NADH alone.
50 c
I
I
NADH
GDH Kinetics in the Polyacrylamide
Gel
The activity of GDH is controlled in a complex manner by a number of effector molecules (38,39). An exhaustive investigation of the kinetic features of the immobilized enzyme was beyond the scope of this study. Nonetheless, it was important to determine the kinetic behavior of the enzyme in the gel and the effects on enzyme activity of NAD and glutamate, the coenzyme and il O 0 ADDITIVE
I
I
I
I
1
2
3
4
CONCENTRATION
(mM)
FIG. 3. Effect of varying NADH and NAD concentrations in the gel-forming solution on the GDH enzymatic activity of the gel. NADH was varied between 0.1 and 2 mM. Higher concentrations of NADH were not tested because they interfered with the process of gel polymerization. NAD was varied between 0.1 and 4 mM. In the case of NAD the solution also contained ADP at a concentration fourfold higher than that of NAD. Each point is the average of 8 to 12 measurements, except the point at 2 mM which is the average of 32 measurements. Bars show the SD.
ADP, ATP, GTP, and ZnC12. The extensive literature on GDH has demonstrated that under appropriate conditions ADP, ATP, GTP, and ZnClz can act as activators of enzymatic activity (reviewed in (24-26)). We tested the effectiveness of each of these four molecules at concentrations up to 36 mM as GDH protectors during immobilization. We found all to be less effective than 2 tIIM NADH alone (data not shown). We then investigated whether these molecules when added to 2 mM NADH increased enzyme protection. Results of these experiments are shown in Fig. 4. ADP, ATP, and GTP in concentrations up to 36 mM did not enhance enzymatic protection above that obtained with NADH alone. ZnC12 reduced the ability of NADH to protect the enzyme. Based on
ADDITIVE
CONCENTRATION
(mt.4)
FIG. 4. Effects of varying concentrations of GTP (O), ATP (0), and ZnClz (A) added to 2 rnM NADH in the gel-forming solutions on the GDH enzymatic activity of the polymer. The molecules tested were added to 2 mM NADH at concentrations that ranged from 0.1 to 2 mM. The other components of the gel-forming solution were held constant at the standard concentrations given in the text. The effects of 2 mM NADH alone are also shown in the figure (m). The effects of added ADP were also tested and they were nearly the same as those of ATP, but they are not shown in the illustration. The points are the average of 4 to 8 individual measurements, except the NADH alone which is an average of 32 measurements. The bars indicate the SD.
ENZYME
ELECTRODE
MEASUREMENT
substrate in the quantitative assay of L-glutamate. In addition to the obvious need to characterize the gel for its use as an analytical tool, kinetic studies also investigate whether the immobilization procedures alter the function of the enzyme. A Lineweaver-Burk plot of the activity of immobilized GDH as a function of NAD concentration is illustrated in Fig. 5. These data were obtained in the presence of 25 mM glutamate, a concentration that saturates enzyme velocity. As shown, the data is best fitted by two intersecting straight lines. The existence of two components in the Lineweaver-Burk demonstrated in Fig. 5 is also characteristic of the activity of the enzyme in solution and has been repeatedly reported in the literature (39,40). The two kinetic states are presumed to arise from the existence in GDH of two distinct nucleotide-binding sites, but the precise molecular mechanism that gives rise to the observed change in kinetics is not understood (40-42). By fitting straight lines to the experimental data, two kinetic domains can be defined one of Kmapp
10
195
OF L-GLUTAMATE
r
01;
’
1
’
2
(L-GLUTAMATE
’
3
’
4
’
5
’
6
CONCENTRATION)-’
I 10 (NAD
I 20 CONCENTRATION)-’
I 30
I 40
I 50
hM-1)
FIG. 5. Lineweaver-Burk plot of the velocity of GDH activity in polymer gels as a function of NAD concentration. The gels were assayed in the presence of 25 mM x&8amate. Just as in solution, two kinetic components are apparent in the plot. They are presumed to arise from the existence of two distinct nucleotide-binding sites in the enzyme (42,43).
’
8
’
9
’
10
(md)
FIG. 6. Lineweaver-Burk plot of the velocity of GDH activity in the polymer gels as a function of L-glutamate concentration. The gels were assayed in the presence of 8 mM NAD.
= 1.14 mM and V,,, = 1.455 pmol/min . mg protein and the other of KmaPP = 0.12 mM and Max = 0.34 pmol/min . mg protein. GDH activity in the gel as a function of L-glutamate concentration assayed in the standard conditions and in the presence of 8 mM NAD is illustrated in a LineweaverBurk plot in Fig. 6. The data were fitted with a single line from which a Kmapp = 0.85 mM * mg protein were and V,,, = 0.98 pmol/min calculated. The kinetic behavior of the enzyme immobilized in the gel, within the few kinetic parameters tested, is not different from that of the enzyme in solution. Immobilization as described here apparently does not alter significantly the function of the active, bound enzyme. Speed of Response of the Polymer
0
’
7
Gel
To measure the response time of the GDH-containing gel to a step change in glutamate concentration we constructed a small chamber that permitted the continuous monitoring of the optical absorbance of a thin, about 70 pm, gel film. A typical continuous time record of the change in absorbance at 340 nm of the gel film following a sudden change in glutamate concentration from 0 to 25 mM is shown in Fig. 7. In the record, the sudden and steady increase in absorbance, indicative of the production of NADH, is nearly simultaneous with the change in glu-
196
KORENBROT,
+Glu
(25
PERRY, AND COPENHAGEN
mM)
o-4 Time
(se4
FIG. 7. Speed of response of thin enzyme gels. The absorbance at 340 nm of the gel produced in the chamber illustrated in Fig. 1 was monitored continuously. The gel was initially bathed with the standard assay solution described in the text. That solution was rapidly exchanged for one containing 25 mM L-glutamate and 8 mM NAD. Within the resolution of the instrumentation, the absorbance of the film changed simultaneously with the change in solution.
tamate concentration and exhibits no detectable latency. The initial rate of change in absorbance is, of course, proportional to glutamate concentration for nonsaturating concentrations. The data indicate that the response of the electrode is indeed rapid, well under 500 ms, and in the experiments presented here the measured rate of response is most likely limited by the frequency bandpass of the recording instrument.
limited by the amount of enzyme present in the gel, since NAD and glutamate are in vast molar excess relative to the enzyme. To improve the analytical value of the gel we investigated the effects of increasing its enzyme content. The activity of gels as a function of their enzyme content is illustrated in Fig. 8. We tested gels that varied in protein content between 0.08 and 8 mg/ml. Enzymatic activity of the gel indeed increases proportionately with its protein content over the range tested, although the relationship is linear only at the lower concentrations of less than about 1 mg/ml. The observed nonlinear dependence of enzyme activity on enzyme concentration is unexpected for enzymes in solution, but is characteristic of immobilized enzymes and is, indeed, the kinetic advantage of immobilized over free enzymes in analytical applications ( 13). The gel specificity, that is, its ability to signal the presence of the substrate of interest and not of other structurally analogous sub-
‘r
Detection, Sensitivity, SpeciJicity,and Stability of the Polymer Gel The detection sensitivity of an enzyme electrode is ultimately limited by the kinetic characteristics of the enzyme it contains. The lowest concentration of glutamate detectable by our gels can be determined, in principle, from the glutamate KmaPP of GDH in the gel by well established theoretical methods ( 13). Calculations show that our electrode should at least detect about 3 PM glutamate. The experimental limit of sensitivity, however, is likely to be given by the signal/noise features of the instrumentation used to measure NADH absorbance and/or fluorescence. In our gels the magnitude of the optical signal is
GDH
CONCENTRATION
(mglgm
gel)
RG. 8. The effect of varying GDH concentration in the gel on its enzymatic activity. GDH concentration was varied between 0.08 and 8 mg per gram of gel by varying its concentration in the gel-forming solution. The other components of the gel-forming solution were held constant at the standard concentrations given in the text. The data points are the average of 4 to 8 measurements, bars are SD. The line was drawn by eye, except at the low concentrations where a straight line was fitted to the points. The nonlinear dependence of enzymatic activity on protein concentration at high concentrations is not observed in solution, but is characteristic of immobilized enzymes.
ENZYME
ELECTRODE
MEASUREMENT
strates, should be entirely defined by the specificity of the enzyme. GDH is highly selective in its deamination reaction for L-glutamate over other substrates (reviewed in (26)). For neurotransmitter studies it is important to establish the selectivity of the GDH in the gel for L-glutamate over structurally similar putative neurotransmitters. Under our standard assay conditions, there was no spectrophotometrically detectable signal in the gel (less than 0.0005 OD/min) in response to the application of up to 435 mM D-ghtamate, 435 mM L-aspartate, 350 mM D-aspartate, and 400 mM N-acetyl-Lhistidine. Since L-glutamate in concentrations of 5 pM produces an easily detectable signal, the response for L-glutamate of the gel exceeds that for D-glutamate, D- and Laspartate, and N-acetyl-L-histidine by at least 1OOO:l. To determine the long-term stability of the electrodes, we produced gels, collected and washed them as usual, and then resuspended them in the normal assay solution containing 8 mM NAD and no glutamate. This suspension was stored at 4°C. The presence of NAD in the storage medium was necessary to maintain activity in the gel over a long term of time. Because NAD is not stable in solution for long, we refreshed the NAD in the gel suspension every 3 to 4 days. At daily intervals aliquots were removed from the gel suspension, exhaustively washed free of NADH, and assayed for their enzymatic activity in the usual manner. We found the gels to be stable, that is, not to lose any appreciable enzymatic activity, for up to 25 days after their production. We did not explore longer time periods. Also, we did not explore the behavior of the gel on repeated use. DISCUSSION
With detailed attention to methodological procedures we have produced hydrophilic and permeant acrylamide polymers that contain covalently immobilized bovine liver
OF
L-GLUTAMATE
197
GDH with preservation of about 50% of the activity of the enzyme. These polymers are the basis of enzyme electrodes to measure L-glutamate that are sensitive, specific, rapid, and stable. The immobilization of GDH described here represents a specific application of a methodological concept pioneered by Adalsteinsson et al. (23). The method developed here can be generalized to the covalent immobilization of other enzymes to produce electrodes for other substrates. To preserve enzymatic activity in the gels several general procedural conditions should be met. First, oxygen should be carefully excluded from the reaction mixtures and all reactions should be carried out at 4°C. Second, the reaction of enzyme with the covalent immobilizing agent, NAS, should be investigated over a range of concentrations to optimize the efficiency of retention while minimizing the potential inactivation of the enzyme. Third, the acrylamide monomers and the catalytic agents in the free radical polymerization of acrylamide should be used at the lowest acceptable concentration consistent with the desired mechanical strength, permeability, and gelling time for the polymer. Fourth, the enzyme should be protected from inactivation by substrates, cofactors, and coenzymes. To measure the concentration of L-glutamate with the GDH enzyme gels there are two reaction products that can be measured, NADH and NH4. The first can be monitored optically by its absorbance or its fluorescence, while the second can be monitored with an ion-specific electrode (13). In the present study we elected to follow photometrically the production of NADH by measuring the absorbance of the gel at 340 nm. Optical methods have a disadvantage when compared to electrochemical ones in that the necessary instrumentation is often more complex. This experimental option is particularly important when considering the application of these enzyme gels to studies of neurotransmitter release in single cells. We wish
198
KORENBROT,
PERRY, AND COPENHAGEN
to point out that optical methods offer a unique advantage over electrochemical ones that justifies the necessary instrumental complexity: The delays imposed by the diffusion of reaction products from the reaction site to the electrode surface are eliminated (13). The response time of the electrode is, therefore, likely to be limited only by the diffusion rate of the substrate through the gel. Indeed, we have shown here the experimental response time of our gels to be rapid, under 500 ms, and likely to be limited by the instrumental frequency response characteristics of the spectrophotometer used. GDH has been used in glutamate-detecting polymers in several previous designs (17-22) and has also been used in solution in assays of L-glutamate (15). The principal advantage of the design described here is the ability to cast electrodes of any desired shape because the gel making solution is fluid for a relatively long and reproducible amount of time. Two previous reports have investigated in detail the kinetic consequences of immobilizing GDH. Barbotin and Breuil (43) immobilized GDH in an albumin film using glutaraldehyde as crosslinker, whereas Julliard et al. (44) bound GDH to a collagen film. The authors found some modification of the control features of the enzyme upon immobilization. The qualitative and quantitative nature of the changes, however, were different in each of the two methods. The allosteric and regulatory features of GDH were not exhaustively examined in the polyacrylamide gels described here. To the extent that they were investigated, the features of the enzyme in the gel were indistinguishable from those in solution. More extensive studies might possibly reveal some kinetic changes. However, for the purpose of analytical investigation the gels described here provide a good basis for reliable and stable enzyme electrodes to be used in neurochemical studies. ACKNOWLEDGMENTS This study was supported by Grants EY01586 (J.I.K.) and EY01869 (D.R.C.) from the NIH and a John S.
Adams award to D.R.C. from Research to Prevent Blindness.
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ENZYME
ELECTRODE
MEASUREMENT
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