1980
Anal. Chem. 1982, 5 4 , 1980-1984
(20) Herman, H. B.; Rechnltz, G. A. Anal. Chlm. Acta 1975, 7 6 , 155. (21) Brandrup, J.; Immergut, E. H. "Polymer Handbook": Interscience: New York, 1966; pp V-17-V-22.
RECEIVED for review March 12,1982. Accepted July 1,1982.
M.E.M. gratefully acknowledges the support of the National Institutes of He&h for this research (GM-2882-01). presented a t the Central Regional Meeting of the American Chemical Society, M i d l a 4 - W June 1982.
Immobilized Xanthine Oxidase Chemically Modified Electrode as a Dual Analytical Sensor Robert M. Ianniello, Thomas J. Lindsay, and Alexander M. Yacynych" Department of Chemistry, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08903
Chemlcally modlfled graphlte electrodes contalnlng covalently lmmoblllzed xanthlne oxidase (E.C. 1.2.3.2) have been employed for the potentlometric and amperometrlc detection of xanthlne. Potasslum hexacyanoferrate(I I I ) Is used as the electron acceptor In the enzyme-catalyzed reactlon and allows for the potentlometrlc and amperometrlc measurement of substrate concentratlon. A potentlometrlc response of -30 to -32 mV/decade is observed wlth logarlthmlc Increase In xanthlne concentration due to the concomttant Increase of the ferrocyanlde/ferrlcyanlde concentration ratlo. I n addltion, the reduced form of the medlator can be electrochemically oxldlzed at 4-03 V vs. Ag/AgCI, yielding a steady-state current directly related to xanthlne concentration. Factors lnfluenclng the response In both modes have been obtained so that optlmum operatlng condltlons in each mode can be elucldated.
The construction of chemically modified electrodes (CME) for the purpose of chemical analysis is a logical consequence of CME research. The advent of covalently immobilized biocatalytic CMEs as potentiometric ( I , Z),and amperometric (3-5) sensors has made the use of these devices very attractive due to their simplicity of operation. In general, the mode of operation of these sensors is quite similar to the well-known membrane coated, species selective transducers. Numerous works concerning the practical and theoretical aspects of operation of potentiometric and amperometric membranecoated electrodes have been reported (6). Most of the mathematically predicted response parameters characteristic of these electrodes are applicable to immobilized enzyme chemically modified electrodes (IECME). However, some important differences in IECMEs (primarily due to the thin enzyme layer) have been pointed out. These differences have been cited ( 5 ) as contributing to the superior response characteristics of amperometric IECMEs when compared to conventional membrane electrodes. An electrode which functions as both an amperometric and potentiometric sensor would be of practical value since the same device could be utilized in two modes of operation. We wish to report the construction and response of the immobilized enzyme chemically modified electrode as a dual (amperometric and potentiometric) electrochemical sensor. Xanthine oxidase (E.C. 1.2.3.2) has been covalently attached to a chemically modified graphite electrode via a carbodiimide linkage. In this system, the substrate (Le., xanthine) is catalytically oxidized in the presence of potassium hexacyanoferrate(II1) according to the reaction 0003-2700/82/0354-1980$01.25/0
xanthine
+ 2Fe(CN)t- + H20
-+ XOD
uric acid
2Fe(CN):-
+ 2H+
When employed as an amperometric sensor, the IECME (poised at +0.3 V vs. Ag/AgCl) consumes hexacyanoferrate(I1) and uric acid produced by the enzymatic reaction. The resulting steady-state current is then related to the initial substrate concentration. The electrode also functions as a potentiometric sensor. The zero-current potential vs. the Ag/AgCl reference electrode is primarily due to the increasing ferro/ferricyanide ratio which develops during the enzymecatalyzed reaction. This allows a unique comparison between amperometric and potentiometric modes of operation using exactly the same electrode. This has not been possible before, because differences in electrode characteristics had to be considered when comparing the two different response modes. The various response characteristics of the electrode in both operating modes have been investigated, and a critical evaluation of the relative merits and drawbacks of the two methods is reported. EXPERIMENTAL SECTION Reagents and Solutions. Xanthine (98-loo%), xanthine oxidase (grade I from buttermilk), peroxidase (type 11),flavin adenine dinucleotide (99%),and o-dianisidine dihydrochloride (purified) were obtained from Sigma Chemical Co. (St. Louis, MO). N-C yclohexyl-N'- (2-morpholinoethyl)carbodiimide-methyl p-toluenesulfonate ("Puriss." grade) was obtained from Tridom Chemical Co. (Hauppauge, NY). Sodium p-(hydroxymercuri)benzoate was obtained from Aldrich Chemical Co. Standard solutions of hydrogen peroxide were prepared by dilution of 30% (v/v) H202 (Fisher Chemical Co.). Concentrations were determined by titration with standardized KMn04 The working buffer consisted of 0.05 M Na+-K+ phosphate (pH 8.0) which contained 2 mM K,Fe(CN),, 1mM sodium salicylate, and 0.005% EDTA. Stuck solutions of xanthine were prepared from the working buffer immediately before use. Purified nitrogen (99.998% minimum purity) was obtained from SOS Gases, Inc. (Middlesex, NS). Graphite electrodes (spectroscopic grade) were obtained from National Carbon Co. and prepared in the manner described previously (2, 5 ) . Distilled, deionized water was used for all solutions. All other chemicals were of reagent grade. Apparatus. Potentiometric measurements were made with a Corning Model 135 pH/ion meter and a Houston Instruments Model 2000 recorder. The reference electrode was a silver/silver chloride (saturated KCl), constructed in-house according to the procedure of Sawyer (7). In particular, a porous glass frit (Vycor, Corning Glass Works) was employed as the reference electrode junction, The low electrolyte flow rate provided by this material greatly reduced the IECME drift. An ECO Model 550 potentiostat (ECO Incorporated, Cambridge, MA) was used to apply a constant potential at a three0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. electrode system for all amperometric measurements. The applied potential was monitored with a B&K Precision Model 283 digital multimeter (Dynascan Cop., Chicago, IL).The IECME, platinum mesh, and Ag/AgCl (saturated KCl) electrodes were employed as the working electrode, auxiliary electrode, and reference electrode, respectively. (3teady-statecurrents were recorded with a Houston Instrumentsi Model 2000 recorder. Currents were calculated from the voltage developed across a precision 10004 resistor. All solutions were kept at 30.0 f 0.1 "C by either immersion in a thermostated bath or use of a water-jacketed cell. Preparation of Xanthine Oxidase Electrodes. Graphite electrodes (5 cm X 0.5 cm) were subjected to the following steps: (a) electrochemical oxidation at +2.2 V vs. SCE in 10% nitric acid for 10 s; (b) radio frequency oxygen plasma treatment at 150 mtorr for 1h; (c) activation of available COOH groups with 0.1 g/mL carbodiimide in pH 5.5 acetate buffer for 45 min; (d) reaction with 50 internationalunits of :wnthine oxidase in 20 mL pH 5.5 acetate buffer at 2 "C for 2 h; (e) scrupulous washing with cold distilled water, 1 M NaCl, and pH 8.0 phosphate buffer. These electrodes were preconditioned and stored in 0.05 M phosphate buffer (pH 8.0) containing 100 pM FAD, 1mM sodium salicylate, and 0.005% EDTA at 2 "C. Assay of Bound Xanthine Oxidase. The total activity of xanthine oxidase bound to electrodes was determined by measuring the hydrogen peroxide produced in 1mM xanthine solution at pH 8.0. Each electrode was placed in 10 mL of oxygen saturated 1 mM xanthine and allowed to react for 5 min in a thermostated shaker bath at 25.0 f 0.1 "C. A 200-pL aliquot of the reaction mixture was mixed with 4 mL of the colorimetric reagent (0.05 mg/mL o-dianisidine, 0.02 mg/mL peroxidase in 0.1 M pH 7 phosphate buffer). The absorbance of the samples was measured at 500 nm after 10 min and compared to that obtained for solutions of known H202concentration. Potentiometric Substrate Determinations. A 13.0-mL solution of the working kiuffer was placed in a thermostated cell and deoxygenated with water-saturated nitrogen for 15 min. The electrodes were immersed in the solution and allowed to reach thermal equilibrium. Various aliquots of the xanthine stock solution (10 mM in working buffer) were added, and the potential change was recorded when steady state was observed (2-5 min). Substrate concentrations were calculated based on the dilution of the aliquot and increaf;eof the final volume of buffer solution. During measurement, stirring was provided by nitrogen bubbling. Amperometric Subst rate Determinations. The electrodes were immersed in 100.0 niL of deoxygenated working buffer and allowed to reach thermal equilibrium. During this time (10-15 min), the potential of the working electrode was stepped to +0.3 V vs. Ag/AgCl reference. The background current was allowed to decay to a steady-state value after which aliquots of the xanthine stock solution were added. The resulting steady-state current which followed brief stirring (5 s) was then recorded in quiescent solution. RESULTS1 AND DISCUSSION Xanthine oxidase is one of the most complex of the oxidase class of flavoenzymes. h i catalytic activity has been attributed to a complex interaction of the substrate with iron, molybdenum, labile sulfides, and flavin adenine dinucleotide at or near the active site. It hELq been proposed (8)that the reaction mechanism follows a sequential "ping pong" pathway. Of particular interest, however, is the fact that the enzyme can utilize a variety of chemical electron acceptors in order to recycle the active site bark to its native state after substrate oxidation. One of these electron acceptors, K3Fe(CN)6,can be employed to form the basis for dual electrochemical detection. The reaction products (Fe(CN)," and uric acid) can be conveniently oxidized at +0.3 V vs. Ag/AgCl with the exclusion of electrochemical xanthine consumption (9). Oxidation of both reaction products should allow for greater sensitivity than previously observed for amperometric IECMEs (5). Concurrently, the increasing ferrocyanide-ferricyanide ratio (in the absence of an applied potential), due to increases in substrate concentration, can be monitored potentiometrically. As Fe(CN)63-/Fe(CN)64-are species com-
54, NO. 12, OCTOBER 1982
1981
300r
> E
v
\
l
I
I
-6
I
!
-5
-4
LOG
I
-3
I
-2
[xanthin4
Flgure 1. Potentiometric response of the xanthine oxidase chemically
modifled electrode in 0.05 M,pH 8.0 phosphate buffer at 30 "C: (A = Xanthine) slope and correlation are -30.1 f 1.4 mvldecade and 0.998, respectively; (0 = xanthine in the presence of 1.5 mglmL p -(hydroxymercuri)benzoate(enzyme inhibitor))slope and correlation are -5.3 f 0.4 mvldecade and 0.975, respectively. prising a reversible redox couple, a potentiometric response of -60 mV/decade increase in substrate concentration is expected a t 30 OC when an ideal redox sensor is used as the indicator electrode. The use of the ferricyanide-ferrocyanide redox system in various potentiometric and amperometric lactate dehydrogenase electrodes (10, 11) has been demonstrated. With the properties in mind, the following response characteristics are presented. The activity of xanthine oxidase, covalently attached to modified graphite as described previously, was determined for three prepared electrodes. The assays were performed on electrodes which were preconditioned for 24 h by soaking in pH 8.0 phosphate buffer containing FAD, salicylate, and EDTA. These conditions serve to stabilize the enzyme (12). It was found that the total mean activity of the tested electrodes was 23.3 f 1.6 nmol mi&. While the enzyme activity appears to be low, it has been demonstrated (13) that collagen membranes containing covalently immobilized glucose oxidase of low activity (16-60 nmol min-l) could be employed to produce sensitive amperometric enzyme electrodes. Buck et al. (14) have demonstrated that conventional potentiometric enzyme-electrodes can be constructed with membranes possessing enzyme activity as low as 15 nmol min-'. The reproducibility of the activity assays in this study (7% relative standard deviation) is indicative of the properties of covalently attached rather than adsorbed enzymes. This property has also been observed for glucose oxidase attached to chemically modified reticulated vitreous carbon (15). In a previous study, the adsorption of xanthine oxidase on graphite electrodes was proved to be negligible when compared to the amount of covalently attached enzyme when the proper washing procedure was used (i.e., 1 M NaCl) (16). Potentiometric Response Characteristics. The potentiometric response of the xanthine oxidase chemically modified electrode to varying xanthine concentration at 30 "C is shown in Figure 1. Response with logarithmic variation of the substrate concentration from 8 X lo4 to 6 X M is observed. The corresponding slope, intercept, and standard error of estimate are -30.1 f 1.4 mV/decade, 134.9 f 5.7 mV, and 4.0 mV, respectively. More detailed response characteristics are listed in Table I. Also shown in Figure 1is the response of the electrode in the presence of p-(hydr0xymercuri)benzoate, a well-known inhibitor of xanthine oxidase (17). As is evident, the presence of the inhibitor drastically attmuates the slope of the potential response to 16% of its original value. It should be noted that inhibition of the enzyme will take place
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
1882
I
Table I. Potentiometric Response Characteristics of the Xanthine Oxidase Chemically Modified Electrode response time,a drift,b reproducibility,c concn, M min mV/min mV 1.5 X lo-' 3.7 x 1 . 2 x 10-4 1.4 X
5.0
279.7
k
h
G 9
2.8
>
-0.2 2.5 2.0
-0.1
300-
Q
E
251.2 f 2.4 225.7 f 1 . 5
v
_I
Change of potential a 98% of steady-state potential. over a 5-min time period. Average potential of five trials standard deviation.
4 c
250-
z
+_
2001
, -6
, -5
LOG
,
,
,
-4
-3
-2
kanthind
Figure 3. Potentiometric response of the xanthine oxidase chemically modified electrode in 0.05 M phosphate buffer at: 25 O C (a),30 O C (A),and 35 O C (U). Slopes at 25, 30, and 35 O C are -32.4 f 2.6, -30.1 f 1.6, and -31.2 f 1.6 mV/decade.
Y >
E
v
-6
-5
-4 LOG
-3
-2
[xanthine]
Figure 2. Effect of pH on the potentiometric response of the xanthine oxidase chemically modified electrode. Phosphate buffers (0.05 M) were used for all Vials: 0 = pH 7.0, 0 = pH 8.0,A = pH 9.0. Slopes at pH 7, 8, and 9 are -12.7 f 1.8, -30.2 f 2.0, and -17.4 f 1.6 mvldecade, respectively. only if the active site is reduced by substrate (in the presence of xanthine). This indicates that the response observed in the absence of inhibitor is determined by the extent of the enzyme catalyzed reaction and not by possible impurities or other components of the sample solutions. It should be noted, however, that the magnitude of the response is 50% less than that expected for a reversible, one-electron process. Stulik (18) has shown previously that graphite electrodes which have been subjected to surface oxidation display diminished potentiometric response to reversible redox species. This has been attributed to the fact that the oxidized surface approaches the structure of graphitic oxide and is therefore no longer an ideal redox sensor. As the electrode constructed in this study has undergone extensive surface oxidation, it was suspected that the diminished response was due to the nonideal character of the modified electrode. The potential response of the xanthine oxidase electrode with different ratios of ferrocyanide-ferricyanide in the absence of xanthine was investigated. Ferricyanide was maintained at constant concentration in all trials (1mM) in order to simulate the enzyme-substrate test conditions. A potentiometric response of -36.4 f 2.4 mV /decade was observed which is quite similar to that obtained in Figure 1. This result shows that the potentiometric response of the enzyme electrode is due to the increasing ferrocyanide-ferricyanide ratio at the electrode surface. The electrode response was observed at different pH values and is illustrated in Figure 2. Optimum response is obtained at pH 8.0. This is in agreement with behavior previously observed for xanthine oxidase covalently attached to glass beads (19). It appears that precise control of pH is needed for optimum potentiometric response.
-6
-5 -4 LOG [xanthin4
-3
-2
Flgure 4. Effect of buffer concentration on the Potentiometric response of the xanthlne oxidase chemically modified electrode. Phosphate buffers (pH 8.0)were used for all trials. = 0.01 M, A = 0.1 M, 0 = 0.5 M. Slopes at 0.01 M, 0.1 M, and 0.5 Mare -30.3 2.0, -26.2 f 1.7, and -11.0 f 1.2 mV/decade, respectively. The effect of temperature on response is illustrated in Figure 3. Electrodes tested at the three reported temperatures demonstrated similar slopes. Within experimental error, the slopes are essentially identical. In addition, temperature did not seem to have any effect on any other response characteristics. One exception is the absolute potential values which vary in the region of linear response by approximately 30 mV. This behavior is similar to that observed for the L-amino acid oxidase IECME (2). The influence of buffer concentration on the potentiometric response is shown in Figure 4. It is demonstrated that a significant response is obtained for buffer concentrations less than 0.5 M. A t 0.5 M, however, a 63% reduction in slope occurs. It is possible that enzyme inhibition due to salt effects (20) occurs at the elevated buffer concentration. It is also likely that FAD dissociation takes place during the measurement at 0.5 M, resulting in decreased enzyme activity in the process, The dissociation of FAD from xanthine oxidase by use of solutions of high salt concentration has been shown previously (16). Analytically, this indicates that buffer concentrations (and storage conditions) in excess of 0.1 M should be avoided. Table I1 is a compilation of the observed slopes of a single electrode over a span of 18 days. The electrode retains 100%
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
1983
Table 11. Operational Stability of the Xanthine Oxidase Chemically Modified Electrode in the Potentiometric Mode slope,a Syx,a,b day mV/decade linear range, M mV 1
5 7 12 18
-30.2 ?: 1.4 -30.1 i: 2.0 -32.4 i 2.6 -22.5 ?: 1.2 -14.6 f 0.7
1x 1x 8x 8x 8x
a Linear regression values. estimate.
10-6-5 X 10-6-5X 10-6-5 x 10-6-1 x
10m6-1X
4.0 8.9 8.9 4.0 2.4
Standard error of the
operational stability of 7 days. Degradation of response occurs after this point with 51% loss of response being observed after 18 days. Xanthine oxidrtre is particularly unstable in solution @ I ) , although it is stablle as a suspension at 2 "C for over 6 months. It appears that immobilization can yield a useful potentiometric electrode with improved stability as compared to the solution-state enzyme. Amperometric Response Characteristics. The dynamic amperometric response of'the xanthine oxidase electrode after addition of substrate (0.49 mM) displayed the following characteristic behavior. A brief response lag occurred after addition of substrate followed by a rapid rise in current. The magnitude of response ici quite large (40 PA), with a dramatic reduction in background current (15 PA) as compared to previously reported amperometric IECMEs (5). The smaller background current is primarily due to the less anodic applied potential employed in this study. In this light, the oxidation of Fe(CN)t- is advantageous compared to HzOzconsumption. In fact, preliminary attempts to measure xanthine concentrations via HzOzoxidation resulted in very low current response. This is primarily the result of simultaneous oxidation of products and xanthine at the applied potential needed to oxidize peroxide. It is possible that the electrochemical consumption of xanthine is faster than the turnover rate of the bound enzyme, resulting in decreased xanthine concentration at the electrode surface. This would result in decreased apparent electrochemical activity due to decreased production of ferrocyanide and uric acid. In addition, the oxidation of xanthine results in adsorption of a diimine product (9),which possibly inhibits the action of xanthine oxidase. In any event, the use of Fe(CN)63-allows one to "fine tune' the amperometric measurement process so that xanthine oxidation is avoided. The response of the electrode at 0.49 mM xanthine before and after the addition of' 1.5 mg/mL p-(hydroxymercuri)benzoate resulted in an 81% reduction in current response over a 5-min time period (95% current reduction occurs over the course of 30 min). This confirms that enzymatic production of products determines the extent of the amperometric signal. This also shows that xanthine oxidation has a negligible contribution to the amperometric response. The steady-state current responses of various concentrations of xanthine were used to construct a response curve for the electrode. This is shown in Figure 5. Linear response is obtained in the xanthine concentration range 6.5 X lo4 to 4.5 X M. The corresponding slope, intercept, and standard error of estimate are 58.6 f 1.3 PAlmM, 1.62 f 1.2 FA, and 5.52 MA,respectively. More detailed response characteristics are given in Table 111. Of interest is the enhanced sensitivity observed for the electrode compared to previously reported IECMEs (5). The electrochemical consumption of both reaction products apparently increases sensitivity. In addition, the electrochemical cycling of ferricyanide at the electrode surface results in the maintenance of an effective constant concentration of the mediator during all amperometric mea-
l
I
I
I
10'~
10-2
banthinel . M
Figure 5. Amperometric response curve for xanthine. Steady-state currents were measured at +0.3 V vs. AgIAgCi.
'01
-a
A A
.3 50. c l
A
z W
[L [L
A
0 3
A
A
A
A
30.
65
05
75
95
PH
Figure 6. Current response of the xanthine oxidase chemically modified electrode at different pH values. Phosphate buffer (0.05 M) was adjusted to the appropriate pH just prior to analysis. [xanthine] = 0.5 mM.
Table 111. Amperometric Response Characteristics of the Xanthine Oxidase Chemically Modified Electrode response reproduciconcn, M time,b min bility,a /*A L O X 10-5 5.0 x 10-4
1.5 x 10-3
1.0
?
0.3
1.8 i 0.5
5.8 i 0.5 13.6 f 0.9 95.5 i: 2.5
a Average current of five trials i standard deviations. Average response time of five trials i: standard deviation.
surements. It is important to realize that all solutions must be oxygen free so that formation of HzOz via O2-enzyme interaction does not reduce the apparent electrochemical activity of the bound enzyme. As compared to the potentiometric characteristics, amperometric measurements result in similar reproducibility with slightly improved linear response range and response time. This would agree with the theoretically predicted behavior of an amperometric enzyme electrode (6). The effect of pH on the current response of the electrode is shown in Figure 6. Increasing current is observed up to pH 8.0, with an observed decrease at pH 8.5. This behavior is in agreement with that of the native enzyme (22). While there appears to be current enhancement at more basic pH values, these currents were not reproducible and did not reach a steady-state value. Currents a t pH 9.2, 9.5, and 10.2 decreased over a 5-min period to 25% of their maximum value. As this is not in agreement with the behavior of the native
1984
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
of an anodic applied potential slightly reduces operational stability. Decreased operational stability as compared to storage stability for amperometric glucose oxidase IECMEs has also been previously reported (4). LITERATURE CITED
c
z
W
w w
I/T x
io3
,
(KO)"
Figure 7. Arrhenius-Van't Hoff plot for the xanthine oxklase chemically modifled electrode. [xanthine] = 0.2 mM.
Table IV. Operational Stability of the Xanthine Oxidase Chemically Modified Electrode in the Amperometric Mode day 1 2 4 1 10 11 currentapA 43.0 44.0 43.5 40.0 29.5 14.5 a
[xanthine] = 0.24 mM in all cases.
enzyme (22), it is possible that hydrolysis of the enzymeelectrode linkage occurs at high pH. This belief is supported by the fact that electrodes tested at pH 10.2 showed no response when later tested at pH 8.0. These results necessitate the use of pH 8.0 for amperometric measurements. The response of the electrode at different temperatures was also examined. An Arrhenius-Van't Hoff plot was constructed and is shown in Figure 7. The general trend of increasing response with increasing temperature, similar to the properties of the free enzyme (12), is observed. However, it appears that two straight lines can be drawn through the data, intersecting at approximately 30 "C. This type of behavior has been observed previously with covalently immobilized L-amino acid oxidase (23) and can be attributed to the presence of multiple, temperature-dependent conformations of the enzyme. The operational stability of the electrode in the amperometric mode was examined over an 11-day period and is presented in Table IV. Identical responses are obtained for 4 days with slight degeneration after 7 days. As compared to the electrode's observed potentiometric stability, the use
Yamamoto, N.: Nagasawa, Y.; Sawai, M.; Sudo, T.; Tsubomura, H. J. Immunol. Methods 1978, 22, 309. Iannlello, R. M.; Yacynych, A. M. Anal. Chim. Acta 1981, 131,123. Kamin, R. A.; Wllson, 0. S. Anal. Chem. 1980, 52, 198. Bourdlllon, C.; Bourgeois; J. P.; Thomas, D. J. Am. Chem. SOC. 1980, 102,4231. Ianniello, R. M.; Yacynych, A. M. Anal. Chem. 1981, 53,2090. Carr, P. W.; Bowers, L. D. I n "Chemical Analysis"; Elving, P. J., Winefordner, J. D., Eds.; Wiley-Interscience: New York, 1980; Vol. 56, pp 206-310 and references therein. Sawyer, D. T.; Roberts, J. L. "Experimental Electrochemistry for Chemists"; Wlley-Interscience: New York, 1974; p 40. Lam, C. F. "Techniques for the Analysis and Modelling of Enzyme KInetic Mechanlsms"; Research Studies Press: England, 1981; pp 255-257. Owens, J. L.; Marsh, H. A,; Dryhurst, G. J. Electroanal. Chem. 1978, 91,231. Shinbo, T.; Sugiura, M.; Kamo, N. Anal. Chem. 1979, 51, 100. Wllliams, D. L.; Doig, A. R.; Korosl, A. Anal. Chem. 1970, 42, 118. Massey, V.; Brumby, P. E.; Komal, H.; Palmer, G. J. Biol. Chem. 1989, 244, 1682. Thevenot, D. R.; Sternberg, R.; Coulet, P. R.; Laurent, J.; Gautheron, D. C. Anal. Chem. 1979, 51, 96. Waiters, R. R.; Johnson, P. A,; Buck, R. P. Anal. Chem. 1980, 52, 1684. Wleck, H. J.; Shea, C.; Yacynych, A. M. Anal. Chim. Acta, in press. Ianniello, R. M.; Lindsay, T. J.; Yacynych, A. M. Anal. Chim. Acta, in press. Guilbauit, G. G.; Kramer, D. N.; Cannon, P. L. Anal. Chem. 1984, 36,
606. Majer, V.; Vesely, J.; Stulik, K. J. Electroanal. Chem. 1973, 45, 113. Coughlan, M. P.; Johnson, D. B. Biochlm. Blophys. Acta 1973, 302, 200. Guilbault, G. G. "Enzymatlc Methods of Analysis"; Perclamon: New York, 1970; p 17. Fried, R.; Fried, L. W. I n "Methods of Enzymatic Analysis"; Bergmeyer, H. U., Ed.; Academlc Press: New York, 1974; Vol. 2, pp 644-649. (22) Greenlee, L.; Handler, P. J. Blol. Chem. 1984, 239, 1090. (23) Guilbault, G. 0.; Lubrano, G. T. Anal. Chim. Acta 1974, 69, 183.
RECEIVED for review May 6, 1982. Accepted June 28, 1982. A.M.Y. thanks Biomedical Research Support Grants, the National Science Foundation (Grant No. CHE 8022237),the National Institutes of Health (Grant No. GM 28125-01) for research support, and Rutgers University for a Junior Faculty Fellowship. R.M.I. acknowledges support of the American Chemical Society, Division of Analytical Chemistry, and the Procter and Gamble Coothrough the award of a Full Year Fellowship. This work was presented, in part, at the 1982 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (paper no. 901, March 1982.
