Proteolytic enzyme modified metal oxide electrodes as potentiometric

Mitre Corporation, 1820 Dolly Madison Blvd., McLean, Virginia 22102. James A. Osborn .... electrode was then heated at 400 °C for 10 min to allow for...
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Anal. Chem. 1086, 58, 140-144

Proteolytic Enzyme Modified Metal Oxide Electrodes as Potentiometric Sensors David C. Roberts* Mitre Corporation, 1820 Dolly Madison Blvd., McLean, Virginia 22102 James A. Osborn and Alexander M. Yacynych* Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903

I n the present work chymotrypsin and trypsln are lmmoblllzed on IrO, and RuO, coated TI electrodes. The pH-sensltlve metal oxlde surface Is used to monltor the enzyme-substrate reaction potentiometrically, as the hydrolysis of the substrates results In a decrease In pH at the electrode surface In the weakly buffered solution. Varlous parameters such as pH, buffer strength, and temperature are used to characterizethe electrodes. A decrease In the response of the electrodes In the presence of Inhibitors Is demonstrated and Is used to suggest a means of determlnlng these substances.

The immobilization of enzymes onto electrochemical sensors to yield potentiometric electrodes has been well documented ( I ) . In general, an enzyme layer is placed at or near the surface of an ion-selective electrode, which senses a change in concentration of a species involved in the enzyme-catalyzed reaction, namely the substrate. Such a response is then related t o the initial concentration of the species of interest. Potentiometric sensors have also been fabricated that sense the presence of species related to the enzyme-substrate reaction, such as coenzymes or inhibitors (I). In the case of reversible inhibitors, a decrease in the expected response of the potentiometric enzyme electrochemical sensor is related to the concentration of the inhibitor. Such a system allows one to use well-understood and relatively inexpensive synthetic substrates to study and quantify the effects of physiologically important enzyme inhibitors. Proteolytic enzymes offer an especially attractive area for the development of potentiometric sensors, since up to this point their potential in this area has been largely untapped. Unlike most other classes of enzymes, many peptidases exhibit a broad specificity, and the resulting electrodes would be applicable to the measurement of many different types of substrates. While certain peptidases are capable of cleaving a wide range of substrates, some of these show several orders of magnitude of variability in activity toward these substrates and are highly specific, in a relative sense, toward certain peptide sequences. This is especially characteristic of intracellular peptidases involved in peptide hormone processing. These enzymes offer exciting prospects for specific and sensitive sensors for individual peptide hormones and their metabolic precursors and products. The recognition that peptides serve as primary information carriers both within the brain and between the brain and many body processes has made peptide research a rapidly expanding field that is highly dependent on state of the art methodology. It has been demonstrated previously that both IrOz and RuOz films formed on a T i support can serve as pH sensors (2,3).Metal electrodes, which have been modified to be pH sensitive, have been used as potentiometric enzyme electrodes by physically entrapping an enzyme through the use of PVC (4,5 ) and by cross-linking with glutaraldehyde (6). These 0003-2700/86/0358-0140$01.50/0

applications have utilized the enzyme urease to create potentiometric sensors selective for the determination of urea. The availability of surface oxide groups has been recognized as a basis for the covalent attachment of enzymes to this type of support via a suitable linking agent (5). Yamamoto et al. covalently attached trypsin to a TiOz coated T i electrode via CNBr creating a potentiometric sensor capable of binding the inhibitor aprotinin (7). Potentiometric enzyme electrodes have also been fabricated by using glass electrodes to detect changes in pH as a result of the substrate reacting in an immobilized enzyme gel layer (8-10). The gel entrapment method provides experimental simplicity and mild conditions of preparation but is limited by increased response and recovery times as a result of decreased mobility of substrate and product molecules in the gel layer. In addition, there is a limitation on the size of the species interacting with the immobilized enzyme. In the present study i t is necessary to provide for the analysis of rather bulky substrates and inhibitors of proteolytic enzymes. As the size of the substrate increases relative to the enzyme, diffusional-related problems increase to a greater degree because the enzymes themselves are immobile. The direct covalent attachment of enzymes to the electrode surface overcomes these problems by establishing a thin layer of enzyme, which allows for greater accessibility of both the enzyme and electrode surface to solution species. The attractiveness of metal oxide electrodes is enhanced due to their mechanical strength as well as ease of preparation and miniaturization as compared to the glass pH electrode. In all cases, however, electrodes prepared in this manner are often sensitive to buffer capacity to some degree. With the above facts in mind, the proteolytic enzymes chymotrypsin and trypsin have been immobilized via cyanuric chloride to the NaOH activated hydroxyl groups of RuOz and IrOz coated T i electrodes. These electrodes responded potentiometrically to specific substrates. The basic response results from a pH change a t the surface of the electrode due to the hydrolysis of the substrate. This response is diminished in the presence of specific reversible inhibitors. The effects of buffer strength, pH, and temperature are used to characterize the use of proteolytic enzymes in the modification of metal oxide electrodes.

EXPERIMENTAL SECTION Apparatus. A Corning Model 135 pH/ion meter was used for

all potentiometric measurements (Corning, Medfield, MA). A single-junction saturated calomel electrode (SCE) served as the reference electrode. A calibrated glass combination electrode was used for pH measurements. The IrOz and RuOz electrodes were constructed from a titanium rod 5 mm in diameter and 15 cm long (Alpha Ventron, Danvers, MA). All solutions were thermostated at 25 f 0.1 O C , unless otherwise stated, with a waterjacketed cell in conjunction with a Blue M constant-temperature shaker bath (Blue M Electric Co., Blue Island, IL). A high-temperature oven, Model 4601, was used in the preparation of the metal oxide electrodes (Hydor Therme Corp., Pennsauken, NJ). 0 1985 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986

Metal chloride solutions were aspirated in the preparation of the metal oxide electrodes with a Jet-Pak spray atomizer of 100-mL capacity and operated with high-purity nitrogen (Spray On Products, Inc., Cleveland, OH). Homogeneous enzyme assays were performed with a Hewlett-Packard diode m a y spectrophotometer, Model 8450 (Hewlett-Packard, Palo Alto, CA). Immobilized and related homogeneous enzyme assays utilized a flow injection analysis system comprised of a Waters Associates chromatography pump, Model 6000A (Waters Associates, Inc., Milford, MA), a Rheodyne syringe-loadingsample injector, Model 7125, equipped with a 20-wL sample loop (Rheodyne, Inc., Cotati, CA), a Kratos Model SF 769 variable-wavelength UV detector (Kratos, Ind., Westwood, NJ), an Omnigraphic 2000 X-Y recorder (Houston Instruments, Austin, TX), and a water bath thermostated with a Lauda constant-temperature immersion birculator, Model MT obtained from Fischer Scientific. Materials. Chymotrypsin (CT) (Type 11,50 IU mg-'), trypsin (TP) (Type 111, 11000 SU mg-l), N-acetyl-L-tyrosineethyl ester (ATEE), N-benzoyl-L-tyrosine ethyl ester (BTEE), N-abenzoyl-L-arginine ethyl ester (BAEE), D-tryptophan methyl ester (TME),and aprotinin (APT)were obtained from Sigma Chemical Co. Ir1nC1,.3H20 (99.9%) and RumCl3.3H2Owere obtained from Aldrich Chemical Co. Cyanuric chloride (purum grade, Tridom Chemical Co., Hauppauge, NY) was used as received. Acetone was distilled from and stored over 4-A molecular sieves. Tris-HC1 buffer (5 mM) was prepared from reagent grade materials. Methanol used in the dissolution of substrates was of spectroscopic grade. A series of Clark-Lubs buffer solutions (11)were used in the pH-response experiments. Distilled-deionized water was used throughout. All other chemicals were of reagent grade. Preparation of Metal Oxide Coated Electrodes. The electrodes were fabricated from segments of Ti rod that were 0.5 cm long and 0.5 cm in diameter with subsequent machining of one end to ensure the removal of any oxide film on the Ti surface as well as to provide a roughened surface. The segments were washed with hexane, distilled-deionizedwater, 6 M HCl, and again with distilled-deionized water with subsequent drying at 110 "C for 1 h. Solutions of IrmC13.3Hz0and RumCl3-3H2O(10 mg/mL) were prepared by dissolving in distilled-deionized water and 2-propanol, respectively. The electrodes were coated, on the machined end, by aspirating the metal chloride solution onto the rotating electrode in 5-5 bursts with intermediate drying accomplished with a heat gun. This was repeated three times. The electrode was then heated at 400 "C for 10 min to allow for partial decomposition of the metal chloride to the metal oxide. The entire procedure was repeated five times after which the electrode was heated for 3 h at 400 "C to complete the decomposition. The electrode was then cooled and sealed to the end of a glass tube with Parafilm, leaving only the coated metal oxide end exposed. This resulted in a geometrical surface area of 0.78 cm2. Electrical contact was maintained with a Cu wire and a few drops of Hg. Preparation of Proteolytic Enzyme Electrodes. The metal oxide electrode, as prepared above, was stirred in 2 M NaOH for 1 h with subsequent rinsing in distilled-deionized water. After drying under vacuum for a period exceeding 10 h, the electrodes were activated by treatment with a saturated anhydrous acetone solution of cyanuric chloride for 1 h. The electrode was subsequently rinsed in anhydrous acetone and dried under vacuum for 1 h. Enzyme immobilization took place in 2 mL of a 25 mg/mL solution of either chymotrypsin or trypsin in pH 7.0 phosphate buffer. The electrodes were treated for 3 h with gentle stirring. The electrodes were then sequentially rinsed with phosphate buffer, 1 M NaCl, and distilled-deionized water before being stored in the appropriate Tris buffer at 4 "C when not in use. Preparation a n d Analysis of Standard Solutions. Standard solutions of the substrates analyzed were prepared as needed by using 20 or 30% methanol to allow dissolution at suitable concentrationsin the working buffer. The working buffer was 5 mM Tham, 0.1 M CaC12,and 0.5 mM NaN, at pH 7.8 for chymotrypsin and pH 7.6 for trypsin. The standard solutions were equilibrated at 25 "C in a constant-temperature bath. The electrode was first equilibrated in a blank solution, containing no substrate, to establish a value for the electrode potential in the blank solution. The electrode was then immersed in the appropriate quiescent substrate solution while its potential was

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monitored for 10 min vs. SCE. The blank potential was then reestablished by immersing the electrode in another portion of blank solution. The temperature of the solutions being measured was maintained at 25 "C. The pH of the solutions being measured was adjusted, using a glass pH electrode, to a value appropriate to the enzyme immobilized just prior to taking the measurements. Immobilized Enzyme Activity Assays. Immobilized chymotrypsin activity was determined by using a method based on that of Hummel(12). The assay utilizes the observation that the hydrolysis products of BTEE absorb more strongly in the UV (256 nm) than does the substrate. The enzyme is typically assayed by adding it to a BTEE solution and measuring the increase in absorbance with time. Knowledge of the extinction coefficient at 256 nm allows for the calculation of units (U)of enzyme activity which is defined as micromoles of BTEE converted to product per minute. The small amount of chymotrypsin in the immobilized preparations prohibited such a straightforward approach. Flow injection analysis (FIA) was used to develop a method of adequate sensitivity. The details of the experimental procedure will be published elsewhere (13). The FIA system involves monitoring the absorbance of a flowing stream of substrate solution, Aliquots of an identical substrate solution, which is continuously incubated with an immobilized enzyme electrode, are injected into this flowing stream. Small changes in the absorbance of the injected solution relative to its initial state are recorded as a positive peak. Increasing peak height with time provides the kinetic data. Homogeneous enzyme assays can likewise be accomplished. Known activities of free enzyme are assayed in this system in order to determine the number of U of enzyme activity per electrode. RESULTS AND DISCUSSION Metal Oxide Coated Ti Electrodes. I r 0 2 and RuOz coated Ti electrodes were both bluish black when immersed in aqueous solution as has been previously observed (2, 3). A series of Clark-Lubs buffers ranging in pH from 3 to 8 were used to establish the pH response of the electrode prior to enzyme immobilization. Each type of electrode responded linearly to pH over the range examined, with response times of approximately 1min, as had been observed previously with IrO, coatings of Ti (2,5). The slopes of the potential response vs. pH for each type of electrode remained virtually constant while varying in terms of absolute potential from day to day. Both the slope and absolute potential readings remained stable over the course of a day during constant use. This behavior is not unlike that of a glass pH electrode that requires routine calibration. A typical IrOz coated electrode gave an average slope of -65.2 f 1.3 mV/(pH unit) with r = 0.999 and a standard error of estimate of 1.6 mV over the course of 5 days. The slope of the RuO, coatings was typically lower with -52.4 f 1.2 mV being a typical value obtained over the course of five days with r = 0.999 and a standard error of estimate of 1.3 mV. The slope of the response of identically prepared electrodes gave consistently similar values in the case of both Ir02 and RuO, coatings. The values, as given, were recorded at 25 "C by using a constant-temperature cell. These values are in agreement with those reported previously (2, 5, 14). C h y m o t r y p s i n Immobilized E n z y m e Electrodes. Chymotrypsin (EC 3.4.21.1) is a proteolytic enzyme secreted into the small intestine by the pancreas in the form of the inactive precursor chymotrypsinogen A (15). Chymotrypsinogen A is activated by trypsin and other autocatalytic interconversions (16). Chymotrypsin hydrolyzes esters and amides of aromatic amino acids, as well as proteins and peptides. In the case of proteins, peptide bonds involving the carboxyl groups of aromatic amino acids such as phenylalanine and tyrosine are readily hydrolyzed. The hydrolytic reactivity of chymotrypsin is in the order: proteins < amides < esters (especially N-substituted tyrosine esters). Two examples of the latter are N-benzoyl-L-tyrosine ethyl ester (BTEE) and N-acetyl-L-tyrosine ethyl ester (ATEE). Esters of tryptophan, methionine, norvaline, norleucine, and also N-benzoyl-L-ar-

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ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986

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Figure 1. Effect of buffer strength on the potentiometric response of a chymotrypsin-modifiedIrO, electrode for Tris concentrations of (0) 0.005 M, (0)0.05 M, and (A)0.5 M at pH 7.8 with 0.1 M CaCI, In each case. Electrode slopes at 0.005, 0.05, and 0.5 M are 126.0, 10.2, and 0.231 mV/decade, respectively, from to lo-' ATEE. The corresponding r values and standard errors of estimate are 0.992 and 8.3 mV, 0.984 and 0.97 mV, and 0.521 and 0.31 mV, respectively.

Figure 2. Potentlometrlc response of a chymotrypsin-modified IrO, electrode at differentpH values: (0) 7.0, (0)7.8, and (A)8.5 (Tris-HCI buffer(5 mM, 0.1 M CaCI,)). Electrode slopes at pH 7.0, 7.8 and 8.5 are 52.4, 93.1, and 145.8 mV/decade ATEE, respectively. The corresponding linear regions, r value, and standard errors of estimate are 1.6 X 104-10-' M, 0.986, and 7.73 mV, 10-3-10-2M, 0.990, and 6.8 mV, and 2.5 X 10-3-10-2 M, 0.992, and 8.0 mV, respectively.

ginine methyl ester (BAME) are hydrolyzed at much slower rates. The stability of chymotrypsin can be increased by the addition of calcium ions resulting in a higher activity (16). In the present work chymotrypsin is catalyzing an ester hydrolysis of the general form

results in the decreased linear range and increased slope observed with increasing p H in Figure 2. The slight buffering is needed to minimize drift in the bulk pH during analysis due to dissolution of COz. It is suggested that working at the fringe of the useful range of the buffer provides a theoretically predicted response while minimizing drift of the electrode due to bulk pH changes. These conditions apply to the analysis of substrates. Steady-state response times of 5-10 min were observed over the range of concentrations examined, with a decrease in response time being observed upon increasing the substrate concentration. These rather long response times reflect the necessity of making measurements in quiescent solutions. Stirring was found to provide both diminished response and irreproducible results as it adversely affects the p H gradient at the electrode surface resulting from the enzymatic reaction. The stability of a typical enzyme electrode was examined in two ways. The effects of time on the absolute potential change for a given concentration and on the change in response vs. concentration of substrate were observed. The potential response of the electrode was found to be virtually constant over a period of 2 weeks when analyzing a 5 mM ATEE solution. The same behavior was observed when measuring the slope of the response vs. concentration of the ATEE curve. After a period of 3.5 weeks had elapsed, the electrode response had decreased by 50% in the 5 mM ATEE solution. Increasing the temperature of an enzymatic reaction typically results in an increase in rate until a point is reached where the enzyme is thermally denatured, leading to an irreversible decrease in activity. It is important to note, though, that this temperature stability is dependent on both the temperature and incubation time. The temperature employed for this work was 25 "C unless otherwise stated. The response at 37 O C was also examined so as to assess the effect of temperature. While the response times of the measurements decreased to less than 5 min at the elevated temperature, for all concentrations examined, the absolute potential change decreased for a given substrate concentration. This same trend was observed by Papariello et al. (9). Initially, the opposite would be expected as the increase in activity escalates the rate of product formation leading to a greater change in pH. On further examination, though, several effects exist which explain the observed response. The pKa of Tris changes from 8.072 a t 25 "C to 7.752 a t 37 "C (17). The buffer capacity of the Tris buffer system consequently increases with temperature. As Figure 1 illustrates, increasing buffer capacity diminishes the response of the electrode. Another buffer system was

RIR2CHCOCH2CHs + HzO RIRzCHC00---+

+ CH3CH20H + H+ (1)

where R1and Rzare positioning groups characteristic of the amino acid derivative. As the reaction proceeds produck build up at the electrode surface resulting in the electrode response based on a change in pH. In the absence of other effects the slope of the response vs. log [ATEE] would be expected to reflect the p H response of the metal oxide electrode. This value would be expected to approach that of the response vs. p H of the metal oxide electrode for complete conversion of substrate to products a t the electrode surface. Typical calibration curves for chymotrypsin immobilized on an IrOzcoated electrode are shown in Figure 1. The pH response of the electrode does not change after enzyme immobilization as was tested by a series of Clark-Lubs buffer solutions in the absence of substrate. The buffer strength, as adjusted by changing the concentration of Tham, has a marked effect on the response of the chymotrypsin-modified electrode, as seen in Figure 1. This behavior is characteristic of a pH-sensitive base sensor. The useful range of the working buffer lies between pH 7 and 9 and suggests an explanation for the trend observed in Figure 2. The initial pH of each substrate solution is adjusted prior to analysis and remains unchanged in the bulk solution during the time interval over which data are taken (e.g., 10 min). At the surface of the electrode, however, the pH changes drastically. The curves in Figure 2 reflect the response of the electrode for initial bulk pH values of 7.0, 7.8, and 8.5. At an initial pH of 7 the useful range of the Tris buffer is exceeded at the onset of each analysis. At p H 7.8, a response of 134.2 mV, calculated as the value of the net potential change obtained at a concentration of 10 mM ATEE, would correspond roughly to a 2-unit change in pH. A response of 12.9 mV at 1.0 mM ATEE corresponds to roughly a 0.2-unit change in pH. In this case, the former measurement has compromised the local buffer capacity while the latter has not. Therefore, the theoretical slope of the response is approached for pH 7 as it is most free of the buffering effect. However at higher p H values the response of the electrode at low substrate concentrations tends to be suppressed relative to that at higher substrate concentrations due to this buffering effect. This

ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986

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employed to determine the relative effect of this change in buffer capacity. The effect of temperature on the buffering agent KHzP04 is less substantial. The pK, of KHzP04 changes from 7.200 at 25 "C to 7.182 at 37 "C (18). The response of chymotrypsin modified metal oxide electrode to 5 mM ATEE was diminished by 55.5 f 4.6 and 53.1 f 4.3% in the Tris and KHzPOl buffer systems, respectively, upon increasing the temperature from 25 to 37 "C. The response of the base sensor will also change with temperature providing a smaller response for a given pH change. In general, diffusion coefficients of species in solution increase ca. 2-3%/"C. This enhanced mobility is observed as a decrease in response time and may also affect the equilibrium of the pH gradient at the electrode surface during analysis. This latter effect could be likened to the effect stirring has on the response. This thermal stirring most probably accounts for the decrease in response with increasing temperature at a given substrate concentration. The slope of the response vs. -log [ATEE] was observed to be unchanged with temperature. In general, there exists a pH of maximum activity for an enzyme under a given set of conditions. Figure 2 illustrates the effect of changing pH on the slope and linear range of the response. The pH at which the maximum net response is observed was also determined. This involved observing the potential change in a 5 mM solution of ATEE after adjusting the pH of the solution analyzed to values from 6.5 to 8.5. Plotting the net potential change in terms of the % maximum response vs. pH resulted in the relationship illustrated in Figure 3. A maximum at pH 7.8 is observed for the chymotrypsin electrode. A second relationship, as redrawn from the data of Goldstein, provides a comparison of the relationship, obtained as described above, with that of the enzyme under homogeneous conditions (19). It is appropriate that the label for the ordinate axis would be % maximum activity in the case of the "free" enzyme. Strictly speaking, Figure 3 is more an illustration than a comparison. The "free" enzyme activity was determined under constant pH conditions by the pH stat method. However, the immobilized enzyme experienced a continuously changing pH reaching a subsequent equilibrium value. These conditions are not readily reproducible in a homogeneous system for such large pH changes. The shift in the pH maximum is not in disagreement with the trend observed by Goldstein for the reasons given above. The shift reflects the fact that the response of the electrode is not solely a function of the activity of the immobilized enzyme as in the work by Goldstein. While the bulk of the work was performed by using the substrate ATEE, the substrate BTEE was also examined. This gave a response with a slope being approximately 50% of that for a typical calibration curve using ATEE under

-3.5

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Flgure 4. Potentiometric response of a chymotrypsin-modified IrOp electrode to BTEE. The slope, linear region, r value, and standard error M, 0.993, and of estimate are 54 mvldecade, 8.3 X 10-4-5 X 2.6 mV, respectively.

Table I. Comparison of Response with Activity of Chymotrypsin-Modified IrOz Electrodes electrode

activity, U"

response, mVb

I I1 I11 IV

5.4 f O.@ 4.4 i 1.2 1.3 i 0.7 0.45 f 0.09

134.2 i 2.3 122.0 i 0.4 38.6 f 0.6 19.8 f 0.3

"A unit (U) of enzyme activity is defined as 1 Fmol of BTEE hydrolyzed/min; each value was multiplied by lo3, [BTEE] = 5 X lo4 M. * [ATEE] = 5 X lo-' M. CValuesresulted from an average of 2-3 assays.

similar conditions as seen in Figure 4. The higher range of concentration examined was limited by the solubility of the substrate. Two possibilities exist to explain this: the pK, of the reaction product benzoyltyrosine (BT) is less than that for acetyltyrosine (AT) and is more easily buffered. Another difference is that while the solutions used for ATEE analysis were 20% methanol, those for BTEE were 30%. This may have provided for adverse conditions pertaining to enzyme activity. As mentioned, work has been done to extend the use of enzyme-modified electrodes to the analysis of species other than substrates, namely inhibitors (1,20). In these cases, an inhibitor concentration was varied while keeping the substrate concentration constant. The decrease in response was then related to the inhibitor concentration. In one case, the change in rate of the enzyme reaction, as monitored amperometrically in the presence of an inhibitor, was used to characterize this dependence (20). In the present work, the steady-state response of the chymotrypsin-modified electrode, at a constant ATEE concentration of 5 mM, was found to be linearly related to the -log of the concentration of the reversible inhibitor D-tryptophan methyl ester (TME) over the range examined (0.5-5.0 mM). The slope, r value, and standard error of estimate were -58.2 mvldecade, 0.996, and 1.5 mV, respectively. Appropriate enantiomeric amino acid esters act as inhibitors of chymotrypsin while not being significantly hydrolyzed (21). In fact, there was no response to TME alone in the absence of substrate. The pH used for these studies was 7.8 as it provided for the greatest net response in absence of inhibitor as shown in Figure 3. The magnitude of this response determines the range over which the inhibitor concentration can be varied while maintaining sufficient sensitivity. It is suggested that conditions that provide for the optimum determination of substrate do not necessarily apply for the determination of the inhibitor.

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LOG [BREEI Flgure 5. Potentiometric response of trypsin-modified (0)IrO, and (0) RuO, electrodes to BAEE at pH 7.6. The slopes, r values and standard errors of estimate for the IrO, and RuO, electrodes are 77 mV/decade, 0.981,and 7.8 mV and 103.1 mvldecade, 0.989,and 7.7 mV, respectively, over the range of 10-3-10-2 M BAEE.

The activity of chymotrypsin immobilized on IrOz electrodes was determined as outlined and related to its response characteristics. Table I contains the data obtained using two electrodes demonstrating normal response (I, 11) and two electrodes whose responses had degraded (111, IV). A direct relationship between the magnitude of activity and the response of the electrode a t a given substrate concentration is indicated by these data. Trypsin Immobilized Enzyme Electrodes. Trypsin was immobilized on both IrOz and RuOz coated T i electrodes as described. While the bulk of the characterization work was done by using the chymotrypsin electrode, the immobilization of trypsin served to illustrate the extension of the idea to more than one enzyme system, the effect of inhibitors on electrode response, and the use of IrOz as well as RuOz coatings. RuO2 coatings have an advantage both in the economics of starting material and safety considerations as pertaining to the toxicity of the starting material. Trypsin functions physiologically in a manner similar to chymotrypsin. It differs mainly in specificity as governed by hydrophobic and charge-charge interactions. The enzyme hydrolyzes bonds in proteins and peptides involving the carboxyl group of a lysine or arginine residue (16). The substrate analyzed with trypsin electrodes was N-benzoyl+ arginine ethyl ester (BAEE). The ester is cleaved and the resulting hydrolysis products decrease the pH near the surface of the electrode. The initial pH of all solutions was 7.6. Typical calibration curves for BAEE as analyzed by trypsin immobilized on both IrOz and RuOz coated Ti electrodes are shown in Figure 5. The slopes of the calibration curves are 77.0 and 103.1 mV/decade for IrOz and R u 0 2 coatings with

r = 0.981 and 0.989 and standard errors of estimate of 7.85 and 7.69 mV, respectively. The pH responses of the IrOz and RuOz coatings were -64.3 and -52.6 mV/(pH unit) with r = 0.999 and 0.999 and standard errors of estimate of 1.38 and 1.32, respectively, prior to enzyme immobilization. A difference in activity of immobilized enzyme layers is, in all likelihood, the primary factor in determining the response of the enzyme electrode to BAEE as demonstrated for the chymotrypsin electrode. The effect on response of aprotinin, a potent reversible inhibitor of trypsin, was examined by using a trypsin-modified RuOz electrode. A 0.1 mg/mL solution of the inhibitor was found to diminish the response of the electrode by 27% in a 5 mM BAEE solution.

Registry No. ATEE, 840-97-1; BTEE, 3483-82-7; BAEE, 971-21-1; TME, 22032-65-1; APT, 9087-70-1; CT, 9004-07-3;TP, 9002-07-7; IrOz, 12030-49-8;RuO,, 12036-10-1;Ti, 7440-32-6; proteinase inhibitor, 37205-61-1; trypsin inhibitor, 9035-81-8. LITERATURE CITED Carr, P. W.; Bowers, L. D. "Immobilized Enzymes in Analytical and Clinical Chemistry"; Why-Interscience: New York, 1980. Ardizzone, S.; Caruguti, A.; Trasati, S. J. Nectroanal. Chem. 1981, 120, 287. Kuhn, A. T.; Mortimer. C. J. J . Nectrochem. SOC. 1973, 120, 231. Alexander, P. W.; Joseph, J. P. Anal. Chim. Acta 1981, 131, 103. Ianniello, R . M.;Yacynych, A. M. Anal. Chim. Acta 1983, 146, 249. Szumlnisky, N. J.; Chen, A. K.; Liu, C. C. Biotechnol. Bioeng. 1984, 2 , 642. Yamamoto, N.; Nagasawa, Y.; Sawai, M.; Sudo, T.; Tsubomura, H. J. Immunol. Methods 1978, 22, 309. Nilsson, H.; Akerlund, A.; Mosbach, K. Biochim, Biophys. Acta 1973, 320, 529. Papariello, G. J.; Mukherji, A.; Shearer, C. Anal. Chem. 1973, 45, 790. Cullen, L. F.; Rusling, J. F.; Schliefer, A,; Papariello, G. J. Anal. Chem. 1874, 4 6 , 1955. Bower, V. E.; Bates, R. G. J. Res. Natl. Bur. Stand. ( U S . ) 1955, 55, 197. Hummelt, B. C. Can. J. Biochim. Physioi. 1959, 3 7 , 1393. Osborn, J. A.; Roberts, 0.C.; Yacynych, A. M., submitted for publication in Anal. Chlm Acta. Gerlscher, H.; Tobias, C. W. "Advance In Electrochemistry and Electrochemical Engineering"; Why-Intersclence: New York, 1981; Vol. 12, p 203. Lehnlnger, A. L. "Principles of Biochemistry"; Worth: New York, 1982; p 113. Bergmeyer, H. U. "Methods of Enzymatic Analysis"; Academic Press: New York, 1974; Vol. 2, pp 1006-1024. Bates, R. G.; Hetzer, H. B. J. Phys. Chem. 1961, 65, 667. Robinson, R. A,; Stokes, R. H. "Electrolyte Solutions"; Academic Press: New York, 1955; Appendix 12.1, p 520. Goidstein, L. Biochemistry 1972, 1 1 , 4072. Guilbault, G. G.; Nanjo, M. Anal. Chim. Acta 1975, 7 8 , 71. Cuatrecasus, P.; Wilchek, M.; Anflnson, C. B. Proc. Natl. Acad. Sci. U.S.A. 1968, 61, 636.

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RECEIVED for review October 18,1984. Resubmitted July 25, 1985. Accepted July 25,1985. The authors thank the National Institutes of Health (GM 33732-02) for research support.