664
Anal. Chem. 1984, 56, 664-667
Kinetic Analysis of Enzyme Electrode Response C. R. Bradley and G. A. Rechnitz* Department of Chemistry, University of Delaware, Newark, Delaware 19711
Two sources of adenoslne deamlnase enzyme are examined to determine their sultablllty for use In the constructlon of an enzyme electrode for adenosine. Klnetlc parameters for each type of enzyme were evaluated both for homogeneous solutions and for the lmmoblllzed enzyme. Results show that enzyme lmmoblllzatlon by BSA-glutaraldehyde cross-llnklng dgnlfkantly alters the kinetic properties. The reaction product Inosine noncompetltlvely lnhiblts the enzyme reactlon and lengthens the response times of the enzyme electrodes, but to a dlfferent extent for the two sources of enzymes studied. The lmpllcatlons of these klnetlc effects for the deslgn of enzyme electrodes are crltlcally examined.
Enzyme electrodes have received a great deal of attention in the analytical literature owing to their specificity, simplicity, and sensitivity in favorable cases. It has been shown that potentiometric enzyme electrodes can also be effectively used to study enzyme kinetics ( I ) . This situation was predicted for large immobilized enzyme activities at the electrode surface by Brady and Carr (2). In this study the kinetics of the adenosine deaminase catalyzed reaction
Adenosine
Inosine
are examined with an ammonia gas-sensing immobilized enzyme electrode a t substrate concentrations both above and below the K , value for the enzyme. Apparent values of the maximum reaction velocity (V-) and the Michaelis-Menten constant (K,) determined for two different sources of the enzyme are compared, and the effects on both electrode response time and reaction kinetics by inhibition by the inosine product are evaluated. Through comparison studies of the kinetic properties for the homogeneous enzyme reaction and the immobilized enzyme electrode case, it will be shown that both product inhibition and electrode response times can be reduced with proper choice of the enzyme source.
EXPERIMENTAL SECTION Apparatus. All potentiometric measurements were made with a Corning Model 12 pH/mV meter in conjunction with a Heath-Schlumberger SR-240strip chart recorder. A Haake Model FS bath with thermostated cells was used to maintain the temperature at 25 "C; a sample volume of 10 mL was used throughout. Orion Model 95-10 ammonia gas-sensing electrodes were used for all potentiometric measurements. Reagents. Analytical grade reagents and deionized, distilled water were used, unless otherwise stated, for preparation of all solutions. Adenosine, adenosine deaminase (type I11 from calf intestine mucosa and type V from bovine spleen; E.C. 3.5.4.4), bovine serum albumin (BSA), glutaraldehyde (Grade I; 25% aqueous solution), inosine, and tris(hydroxymethy1)aminomethane (Tris) were purchased from Sigma Chemical Co., St. Louis, MO. Procedures. Construction of Enzyme Electrodes. Adenosine enzyme electrodes were constructed by immobilizing 5 pL (>1 0003-2700/84/0356-0664$01.50/0
IU) of adenosine deaminase at the electrode surface. The immobilization was carried out by combining 5 pL of adenosine deaminase enzyme and 5 r L of a 15% BSA solution in working buffer (0.2 M Tris-HC1; 0.001 M ethylenediaminetetraacetic acid at pH 9.0) on the gas-permeable Teflon membrane of the ammonia sensor and mixing thoroughly with a small glass rod. Then 5 pL of the 25% glutaraldehyde was added and the solutions were mixed. The prepared membrane was allowed to dry in air for 20 min, soaked in water for 15 min and then for 15 min in a 0.1 M glycine solution, and allowed to stand overnight in the working buffer at 4 OC. Similar procedures have been used for immobilization of this enzyme (I)as well as for urease (3). The internal electrode filling solution used throughout this study consisted of 0.17 M NaCl and 0.03 M NH4Cl. Determination of Immobilized Kinetic Parameters. A pH profie of both types of adenosine deaminase enzyme was obtained and a calibration curve constructed at the optimum pH value. Apparent K , and V,, values for the enzyme electrode were determined by a plot of the data from initial rate measurements in a double-reciprocal form according to Lineweaver and Burke (4). Substrate concentrations for rate measurements were chosen so as to fall in the working linear range of the electrode, both above and below the K , value of the enzyme. The effect of product inhibition by inosine on both response time and the previously determined kinetic parameters was investigated by varying initial M. Lifetime studies inosine concentration from zero to 2 X of the enzyme electrodes were also carried out. Determination of Homogeneous Kinetic Parameters. A pH profile of both types of adenosine deaminase enzyme in solution was obtained and a calibration curve constructed at the optimum pH value. Apparent K , and V,, values were obtained by using the same procedure as for the immobilized case. Inhibition studies were also performed as in the immobilized procedure. In each set of measurements for both homogeneous and immobilized experiments at least three electrodes were employed to check the reproducibility of the results and to ensure that the results obtained were not influenced by electrode bias.
RESULTS AND DISCUSSION Enzyme Electrode Response Characteristics and Kinetic Parameters. The adenosine deaminase enzymes obtained from calf intestine mucosa (type 111)and bovine spleen (type V) differ in specific activity. For meaningful analytical purposes, it is necessary that experiments be carried out under conditions where the amount of enzyme immobilized is not rate limiting. Examination of Figure 1 reveals that both type I11 and type V enzymes cause maximum reaction velocity to occur when less than one unit is immobilized, any additional enzyme causes no further change in velocity. For this study, 5 p L of enzyme was chosen so as to maintain a constant volume of enzyme and cross-linking reagents for each electrode. This also allows for maximum reproducibility of the enzyme layer thickness from one electrode to another. This point is important in comparison studies since it is necessary to keep diffusion time as a constant instead of a variable that must be taken into account when analyzing results. Typical response curves to adenosine of the resulting enzyme electrodes both show a slope of 54 mV per concentration decade with a linear range extending from 3.5 X 10" M to 1.6 M adenosine (at pH 9.0 and 25 "C). The limit of X detection was found to be approximately 8.3 X lo4 M. The pH optimum for both types of enzymes was found (Figure 2) to be 9.0, which agrees with the literature (5).Table I shows 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56,NO. 4,APRIL 1984
665
Table I. Enzyme Electrode Response as Function of Time day 1 4
10 13
slope, limitof mV/ detection, M decade
linear range, M 3.47 X 8.51 X 8.51 X 8.51 X
8.30 X 9.53 X 1.04 X 10‘’ 1.08 X lo-’
1 0 - 5 - 1 . 6X 10’5-1.6 X 10”-1.6 X 10-’-1.6 x 10.’
54.3 52.7 53.0 52.2
n
> E
Y
0.5
1.5 ENZYME UNiTS
2.5
w
a
Flgure 1. Plot of relative velocity vs. enzyme activity in units for immobilized type 111 (0)and type V (A)adenosine deaminase enzyme. One enzyme unit is the amount of enzyme which deaminates 1 Fmol of substrate/min at 25 ‘C. 5.0 X M substrate. 4
12
20
RESPONSE TIME(min)
Flgure 3. Effect of inosine concentration on type II I enzyme electrode response time. TR = 5 min, 7 min, 12 mln, and 14 min for 0 (A),2 M (0) inosine, respecX lo-‘ M (O), 2X M (A), and 2 X tively. Concentration of adenosine was -3 X
M.
CI
> E w
Y
6
0.25
-i 7.5
4
I
12 RESPONSE TIME (rnin)
I 20
Flgure 4. Effect of Inosine concentration on type V enzyme electrode response time. T , = 8 min, 10 min, 18 min, and 25 min for 0 (A), 2X M (O), 2X M (A),and 2 X M (0)inosine, reM. spectively. Concentration of adenosine was -3 X
8.5
9.5
PH
Figure 2. pH profile for immobilized type I11 (0) and type V (A) adenosine deaminase enzyme. 5.0 X lo-‘ M substrate.
that the immobilized enzyme electrode retains its response properties for a t least 13 days with only a slight decrease in calibration curve slope. Kinetic parameters for the two types of enzyme were found to be significantly different. Lineweaver-Burke reciprocal plots for immobilized adenosine deaminase enzyme yielded apparent K , and V,, values of 3.20 X lod4M (f0.38) and 85.2 mV/min (f2.3) and 4.51 X M (*0.53) and 60.0
mV/min (f3.2) for type I11 and type V enzymes, respectively. These results and the calibration data indicate that the immobilized enzyme electrodes are functioning satisfactorily from the analytical point of view both above and below the K , values of the two enzyme types studied as predicted by Brady and Carr (2). The effect of adding the reaction product inosine to the initial measurement solution is striking. The response time of the type I11 enzyme electrode to a low level of adenosine ( 3X M) was found to increase from approximately 5 rnin with no inosine initially present to 14 rnin with an inosine concentration equal to 2 X M (Figure 3). For purposes of this study, the response time is simply defined as the time necessary for the electrode to reach 95% of its steady-state potential. The effect of inosine on the response time of the type V enzyme electrode was even greater than that on the type I11 enzyme electrode, with response time increasing from 8 min with no initial inosine to 25 min with 2 X M inosine
-
666
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Table 11. Apparent Kinetic Parameters from Lineweaver-Burke Plots for Immobilized Enzymes type I11 enzyme Km,M
vrnax, mV/ min
3.25 X 2.94 x 10-4 3.07 X l o w 4 3.30 X
86.0 74.5 66.9 52.5
[inosine], M 0 2 x 10-4 2X 2X
type V enzyme Vmaw mV/ min
K,,M 4.60 X 4.92 x 1 0 - 4 4.28 X 5.32 X
60.5 56.9 44.8 18.2
d
1.o
I 2.0 ENZYME UNITS
3.0
Flgure 8. Plot of relative velocity vs. enzyme activlty in units for M homogeneous type V adenosine deaminase enzyme. 5.0 X
substrate. in the construction of the corresponding immobilized enzyme electrodes. Calibration curves constructed from measurements taken homogeneously with 1.0 activity units of type I11 enzyme gave slopes of 55.7 mV/decade of adenosine concentration with a linear range extending from 1.5 X M to 8.7 X M, smaller than that of the immobilized case. At adenosine concentrations greater than 8.7 X M the response time of the electrode increased from 15 to about 30 min with only a small change in potential. Similar work with type V enzyme using 3 units of adenosine deaminase showed a slope of 57 mV/decade with a linear range extending from 1.5 x M to only 3.8 X M, significantly smaller than that of the immobilized case. Apparent K , and V,,, values obtained in homogeneous experiments for both types of enzyme were found to be 9.77 X M (f0.24) and 128.7 mV/min (* 1.8) and 9.05 X and (3~0.30) and 91.0 mV/min (k2.7) for type 111 and type V enzymes, respectively. Comparing the immobilized results to the homogeneous results for the type I11 enzyme shows that the apparent K , of the immobilized case is approximately three times greater than the apparent K , value of the homogeneous case; this implies that the enzyme has greater affinity for the substrate when the enzyme is free in solution as opposed to being confined in a cross-link entrapment. The apprent V,, value of the homogeneous case was also found to be greater than the apparent V,, value of the immobilized case. For the type V enzyme the apparent K , value for the immobilized cases was more than four times greater than the apparent K, value for the homogeneous case. The apparent V,, of the type V enzyme in the homogeneous study was also found to be greater than that found when the enzyme was immobilized. Thus, the qualitative trends exhibited by both type I11 and type V adenosine deaminase enzymes are similar, but the type I11 enzyme has moderately more attractive apparent K, and V,, values from the analytical viewpoint. In the homogeneous inhibition study, the results for both type I11 and type V enzyme (Table 111) showed a large decrease in apparent V,, with increasing inosine concentration. This decrease in apparent V,, was of a greater proportion for the homogeneous case than for the immobilized case, perhaps because the inosine has easier access to the homogeneous enzyme than the immobilized enzyme. For the homogeneous study, the inosine produces a change in apparent K , value
c 0.2
0.6
1.o
E N Z Y M E UNITS
Flgure 5. Plot of relative velocity vs. enzyme activity in units for homogeneous type I I I adenosine deaminase enzyme. 5.0 X 10-6 M
substrate. present (Figure 4). It should be kept in mind that the inosine concentration is truly zero only at time zero. When the enzyme electrode is exposed to the sample solution, some substrate consumption to generate inosine is inevitable. An earlier study has shown that such substrate conversion can be significant under certain operating conditions (1). The relative effect of inosine inhibition on the apparent kinetic parameters K, and V,, is shown in Table I1 for both type I11 and type V adenosine deaminase enzyme. These results show a decrease in apparent V,, with increasing inosine concentration with little or no change in apparent K, value of the enzyme. Inosine, therefore, does not appreciably affect the affiiity adenosine deaminase has for adenosine, but acts as a noncompetitive inhibitor to slow the reaction. An unexpected finding is that the extent of the decrease of apparent V,, is dependent upon the type of enzyme examined. For the highest inosine concentration, the apparent V,, value of the type I11 enzyme electrode decreased to 61% of its original value while the apparent V, of the type V enzyme electrode retained only 30% of its uninhibited value. This suggests that type I11 adenosine deaminase enzyme would be a much better selection than the type V for construction of the enzyme electrode since the K, is lower (higher affinity) and the V,, higher for the type I11 enzyme, and the type V enzyme also is subject to a greater inhibitory effect by the reaction product. Homogeneous Studies. Reaction rate measurements carried out with the enzyme in homogeneous solution provide an additional means of obtaining data relevant to enzyme electrode studies. These homogeneous measurements were carried out with high levels of type I11 (Figure 5) or type V (Figure 6) enzyme in order to approximate the levels employed
667
Anal. Chem. 1984, 56,667-671
Table 111. Apparent Kinetic Parameters for Homogeneous Enzyme Studies type V enzyme type I11 enzyme
-Vmax, _
[inosine], M
Km, M
mV/ min
128.7 9.77 h 116.0 2x 9.33 x 2 x 10-3 1.30 x 10-4 110.0 69.2 2X 2.41 X 1 0 - 4
0
I .
Vrnax,
K,,
M
mV/
min
9.05 X lo-’ 91.0 1.15 x 60.5 8.80 x 10-5 37.7 7.16 X lo-’ 12.1
for each type of enzyme but in an opposite direction for each. The type I11 enzyme experiences an increase in apparent K , with increasing inosine concentration, while the type V enzyme shows a slight decrease in apparent K , at high inosine concentration. The most dramatic homogeneous effect is that of increasing inosine concentration upon the V,, values, however. For the type I11 enzyme, apparent V, is reduced by nearly a factor of 2 but, for the type V enzyme, apparent V,, decreases more than 7-fold. Such product inhibition reveals itself through longer response times in the immobilized enzyme electrodes. The results obtained in this study show that the potentiometric ammonia gas-sensing enzyme electrode does exhibit linear responses to substrate concentrations both above and below the K , value of the adenosine deaminase enzyme when sufficient enzyme is immobilized at the electrode surface. The BSA-glutaraldehyde cross-link provides for stabilization of the enzyme activity as shown by the observed electrode lifetime. Comparison of the homogeneous kinetic parameters with those obtained from the immobilized study reveals significant changes in the kinetic properties of the enzyme when
it is immobilized, possibly resulting from conformational changes in the enzyme upon exposure to BSA and glutaraldehyde. The magnitude of the effect of addition of inosine on apparent K , and V,, depends upon whether the enzyme is immobilized or free in solution. The apparent K, for the immobilized enzyme remained essentially constant upon addition of inosine, while the apparent K,,, for the homogeneous enzyme did show some variation but in opposite direction for the type I11 and V enzymes. In the construction of enzyme electrodes, it is desirable to obtain the highest V,, (fastest rate) and lowest K , (greatest affinity) values possible. From the results of this study carried out at high enzyme levels it is apparent that type I11 adenosine deaminase would be the best choice for the construction of an immobilized enzyme electrode both from the point of view of apparent K , and V, values and from the less pronounced product inhibition effect on the type I11 enzyme compared to the Type V enzyme. Even in the absence of initial inosine, type I11 enzyme electrodes have faster response times than corresponding electrodes prepared with type V enzyme. Registry No. Adenosine deaminase, 9026-93-1; adenosine, 58-61-7.
LITERATURE CITED (1) (2) (3) (4) (5)
Arnold, M. A.; Rechnitz, G. A. Anal. Chem. 1982, 5 4 , 2315-2317. Brady, J. E.; Carr, P. W. Anal. Chem. 1980, 52, 977-980. Mascini, M.; Guilbauk, G. G. Anal. Chem. 1977, 49, 795-798. Lineweaver, H.; Bruke, D. J . Am. Chem. SOC. 1934, 56, 658-666. Deng, I.; Enke, C. Anal. Chem. 1980, 52, 1937-1940.
RECEIVED for review October 5,1983.
Accepted December 23, 1983. We gratefully acknowledge the support of NSF Grant CHE-8025625.
Ferrocene-Mediated Enzyme Electrode for Amperometric Determination of Glucose Anthony E. G. Cass,’ Graham Davis, Graeme D. Francis, and H. Allen 0. Hill*
Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom William J. Aston, I. John Higgins, Elliot V. Plotkin, Lesley D. L. Scott, and Anthony P. F. Turner
Biotechnology Centre, Cranfield Institute of Technology, Bedford MK43 OAL, United Kingdom
An amperometric enzyme electrode for the analysls of glucose Is described. The electrode uses a substltuted ferrlcinlum ion as a mediator of electron transfer between Immoblilzed glucose oxldase and a graphlte electrode. A llnear current response, proportional to the glucose concentratlon over a range commonly found in dlabetlc blood samples (1-30 mM), Is observed. Data are presented on the influence of oxygen, pH, and temperature upon the electrode. Results with cllnlcal plasma and whole blood samples show good agreement wlth a standard method of analysls.
In the condition diabetes mellitus, the determination of blood glucose levels rapidly, conventiently, precisely, and Present address: Centre for Biotechnology, Imperial College, London SW7 2AZ, UK. 0003-2700/84/0356-0667$01.50/0
economically is important for its diagnosis and effective management. The routine analysis of glucose in a variety of physiological fluids is one of the most frequent operations in a clinical chemistry laboratory. Many protocols exist for glucose analysis and have been recently reviewed (1). Those most commonly employed, at present, are based upon the use of the enzyme glucose oxidase to catalyze the following reaction: glucose
-
+ O2
gluconolactone
+ H202
(1)
Though the product of the reaction, hydrogen peroxide, may be determined by a number of methods, potentiostatically controlled electrochemical oxidation at a platinum electrode has attracted the most interest. This combination of enzymatic catalysis and current measurement at a controlled potential is a feature of the amperometric enzyme electrode. These devices have been reviewed by Carr and Bowers (2). Despite the emphasis on amperometric determination of 0 1984 American Chemical Society
