J . Phys. Chem. 1993,97, 9060-9063
9060
Kinetics of the Peroxidase-Oxidase Reaction with Immobilized Enzyme Linda Cook, Raima Larter,' Peidong Shen, and Torben Geest Department of Chemistry, Indiana University Purdue University Indianapolis (IUPUI), 402 N . Blackford Sr., Indianapolis, Indiana 46202-3274 Received: April 8. 1993; In Final Form: May 21, I993
Reaction rate studies of the peroxidase-oxidase (PO) reaction were conducted utilizing horseradish peroxidase (HRP) immobilized in cross-linked bovine serum albumin (BSA), an inert protein matrix. It was found that the membrane potential was a valid indicator of the extent of reaction and could be used to follow the kinetics. The rate of reaction in the immobilized system was determined to be approximately 10 times slower than with free enzyme, a result consistent with well-accepted theories of immobilized enzyme kinetics. The measurement of membrane potential thus provides an alternative method to the established spectrophotometric and oxygenselective electrode techniques for monitoring the PO reaction in vitro. In addition, preliminary evidence of nonlinear behavior in the form of damped oscillations was found. The sawtooth shape and period (1-2 min) of the oscillations observed are similar to those seen with free enzyme, but the amplitude was found to be 10-25 times larger. Since some HRP is found to be immobilized in the cell wall in vivo, these observations lend tentative support to the possibility that in vivo oscillations in the PO reaction might also occur.
Introduction The oxidation of reduced nicotinamide adenine dinucleotide (NADH) by molecular oxygen is an enzyme-catalyzed reaction which occurs in vivo in woody plants as the first step in the biosynthesis of lignin.' The overall reaction is NADH
+ 0, + 2H'
-
HRP
NAD'
+ 2H,O
In 1965, it was discovered that this reaction (known as the peroxidase-oxidase, or PO, reaction) occurs in vitro via damped oscillations when peroxidase enzyme extracted from horseradish (HRP)is used as the catalyst? Later investigations revealed that the damped oscillations became sustained when NADH was regenerated either by a second enzyme-catalyzed reaction3or by infusion using a syringe pump.4 In 1977, chaotic oscillations in this reaction were first observed;S recently, a period-doubling sequence of bifurcations was found to be one route by which chaotic behavior arises in this systema6 For these reasons and others, the PO and related reactions have been the object of study of a number of investigators in recent years.' Whether the in vitro observations of nonlinear behavior have applicability to the in vivo behavior of the PO reaction remains unknown. Important differences between the in vitro conditions and the probable in vivo conditions are known to exist; however, it is not known whether any of these differences are crucial in determiningthe existenceof nonlinear behavior such as oscillations and chaos. One difference involves the fact that in horseradish the peroxidase enzyme is bound, i.e. immobilized, in the matrix of the cell wa11lJ while the typical in vitro for the study of nonlinear behavior in the PO reaction have utilized free enzyme. It is well-known that immobilization of enzymes can have an effect on the kinetic behavior of enzyme-catalyzed reactions.9 One reason for this is the fact that substrate must diffuse through the immobilization matrix to the enzyme site, and diffusion through such a matrix is typically slower than through aqueous solution, especially if the latter is well-stirred as is the case in prior studies of the PO reaction. Another reason the rate of reaction differs for immobilized as opposed to free enzymes is that the binding of the enzyme macromolecule to the immobilization matrix can distort the resting conformation of the enzyme, possibly changing access to the active site. Finally, enzyme reactions which are pH-dependent will be affected by a local pH which may differ from that in the bulk. It is not unusual 0022-3654/93/2097-9060%04.00/0
to see a 10-fold decrease in the rate of reaction for immobilized enzymes due to these effectsag For these reasons, weinvestigated thekineticsof thePO reaction using immobilized HRP, paying particular attention to the possibility of nonlinear behavior. The ultimate goal of our research is to find answers to the followingquestions: do oscillationsoccur in vivo in the PO reaction, and are biochemical oscillatory reactions, in general, the fundamental basis of biologicalrhythms? We report here preliminary evidence that oscillations occur in the PO reaction when the enzyme is immobilized; this evidence lends support to the idea that oscillatory enzyme-catalyzed reactions may, in fact, be the fundamental basis for biological rhythms since most enzymes are found immobilizedin membranes in vivo and nearly all important biochemical reactions are enzyme catalyzed. A secondary question addressed here is whether measurementsofreaction ratemade external to theimmobilization matrix can be used as a probe to follow the reaction as it proceeds inside the matrix. The matrices we used were cast in the form of membranes, and one external measurement we have investigated is the membrane potential. Since the PO reaction involves charged species and the immobilization matrix is, itself, charged, the membrane potential is nonzero. We report here evidence that the membrane potential varies with the extent of reaction, making this property an excellent external probe of the reaction occurring within the membrane. Experimental Section Membranes were produced using the cross-linking method of Thomas and Broun.lo HRP (immunoassay grade, BoehringerMannheim) and 55-60mgof bovine serum albumin (BSA, Fluka) were dissolved in 0.6 mL of 0.02 M phosphate buffer (pH 7.0). A 0.4-mL sample of 2.5% glutaraldehyde (in 0.02 M phosphate buffer) was then added to produce an HRP solution of concentration 15-16 mg/mL. After allowing this solution to thicken at room temperature for 4-6 h, it was spread onto a plane glass surface, held at 5 "C until completely solidified and then soaked and rinsed in distilled water to remove the membrane from the glass. The resulting membranes (approximately 3.5 cm in diameter and < 1.O mm in thickness) had an enzyme loading of about 0.8 mg/cm2 (calculated from the weighed amount of enzyme and the final measured diameter of the circular membrane). The membrane was then immersed in a 10 mg/mL glycine solution to block the function of the remaining free glutaraldehyde and 0 1993 American Chemical Society
The Journal of Physical Chemistry, Vol. 97, No. 35, 1993 9061
Kinetics of the Peroxidase-Oxidase Reaction Oxygen Elec
NADH,
@-
a
30-
I
pwentlal(mV) ,
I
Membrane
b
0 , 0
I
1
2
3
NADH (mM) Figure 2. Effect of NADH concentration on measured potential. Circles are for HRP-loaded membrane; [HRP] = 16.5 mg/mL resulting in 0.86 mg/cmz in membrane. Squares are for BSA-only membrane. Transport cell contained 0.001M sodium acetate buffer (pH = 5.0), 25.0 pM 2,4dichlorophenol, and 0.1 pM methylene blue. Solutions were precquilibrated with air prior to injection into the cell. NADH injected into one cell compartment only; [Oz]is monitored in this same side. Temperature is 23 OC.
t-------Sccm---------( Figure 1. (a) Schematic diagram of transport cell. Cell is constructed of plexiglasswith Teflon stirrers. The inner dimensionsof the cylindrical cell are 2.5 cm in diameter by 5.5 cm long. The volume of each compartment is 10 cm3. Calomel electrodes are used to measure membrane potential, and an oxygen-selective electrode is used to measure dissolved [Oz](b) Side view of plexiglass membrane holder that separates the cell compartments. Area of exposed membrane surface is 0.78cmz.
.
held at 5 OC until used. Some membranes were prepared without HRP but in an otherwise identical manner and used as blanks in the rate determinations. The HRP was found to be firmly immobilized by this technique. Leakage of HRP out of the membrane was ruled out by monitoring the absorbance at 403 nm of a solution containing a sample of membrane; no change in absorbance was seen. The membranes were mounted in a plexiglass transport cell shown schematically in Figure 1. The volume of each cell compartment was 10mL, well stirred withmagneticTeflon stirrers and at an approximately constant temperature of 23 OC. Since a continuous flow of oxygen into the reaction medium is desirable, the cells were not designed to be airtight; oxygen thus enters the solution from the atmosphere at a relatively constant rate when stirring is adequate. Sodium acetate buffer (pH 5.0) containing 2,4-dichlorophenol (Aldrich) and methylene blue (Sigma) was pumped into both sides of the transport cell. The solutions used in the kinetic studies contained dissolved oxygen due to equilibration with the atmosphere prior to injection into the cell. In some experiments, oxygen was partially purged from the solution by bubbling pure nitrogen through the solution prior to injection into the cell; bubbling of nitrogen continued throughout the experiment in which damped oscillations were observed. In all cases, the dissolved oxygen concentration was monitored with a precalibrated Clark oxygen-selectiveelectrode (from Microelectrodes, Inc.) Disodium-NADH (Boehringer Mannheim) solutions were injected into one side of the transport cell. NADH solutions were prepared by dissolving small amounts (about 20 mg) of the solid in approximately 1 mL of distilled water (pH 7) no more than 1 h prior to the experiment; this solution was injected into one side of the cell with a small syringe, and the final concentration was calculated using the known solution volume. The oxygen electrodewas used to monitor the decrease in dissolved oxygen concentrationin this side of thecell. Two identicalcalomel electrodes (Radiometer) connected to a Keithley 617 voltmeter were used to monitor the membrane potential.
NADH (IDMI
Figure 3. Calibration curve. The membrane potential is a linear function of [NADH] below approximately 1.5 mM. HRP-loaded membrane (0.80 mg/cm2) was used in transport cell with other conditions as given in Figure 2.
RMultS
Figure 2 shows typical values of the membrane potential as a function of initial NADH concentration for both an HRP-loaded membrane and an otherwise identical blank (HRP-free) membrane. These measurements are the initial potential across the membrane, since, in both cases, the potential will decrease over time as NADH is consumed. The presence of enzyme in the membrane is seen to more than double the measured membrane potential, an effect seen reproducibly for several pairs of membranes. The near linearity of the data shown in Figure 2 suggests that the measured potential can serve as a calibration curve for [NADH] and, hence, the extent of reaction since NADH is consumed by the reaction. Figure 3 shows an identical measurement for another HRP-loaded membrane for which subsequent measurements of reaction rate were carried out. It can be seen that the potential rises nearly linearly with [NADH] up to about 1.5 mM; the slope of this line is 10 mV/mM, giving a conversion factor which can be used in the analysis of the rate data shown in Figure 4. In this sequence of experiments, the oxygen concentration was monitored using the oxygen-selective electrode as the oxidation of NADH proceeded (see Figure 4a).
9062
Cook et al.
The Journal of Physical Chemistry, Vol. 97, No. 35, 1993 180
,
a
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6
200
0
400
0
600
800
lo00
1
200
,
I
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2
3
:
4
:
5
.
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7
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8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 Time (minutes)
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Figure 5. Damped oscillationsfor immobilized HRP membrane. Here, initial [Oz] = 50 pM, which was achieved by bubbling dry NZ(g) into the solution prior to injection into the cell; bubbling of Nz(g) into the solution prior to injection into the cell; bubbling of N2(g) continued throughout the experiment shown. Initial [NADH] = 1.0 mM. Otherwise, same conditions as in Figure 3.
TABLE I: Comparison of Measured Reaction Rates
potential CmV) l5
method AAbs at 340 nm A[O2] Amembrane potential AAbs at 340 nm
rate (M s-') 0.7 X l e 7 1.2 X l P 7
ref this work this work this work 12 13 13
I immobilized enzyme free enzyme
NO21
AAbs at 340 nm (DCP added)
0
200
400
600
800
lo00 1
t h e (sec.)
301-----1
time (sec) Figure 4. Rate studies for immobilized HRP membrane. (a) [ 0 2 ] vs time. The same HRP-loaded membrane described in Figure 3 was used. Initial [NADH] = 1.1 mM. (b) Membrane potential vs time. (c) Rate of oxygen leaking into cell. Here, no NADH added; solution purged of dissolved oxygen prior to injection into the cell.
A simultaneous decrease in potential is also observed (see Figure 4b). This latter measurement can be converted to a decrease in [NADH] using the above conversion factor. The slope of Figure 4b is 4.001 8 mV/s, which thus corresponds to a rate of decrease of [NADH] of 1.8 X l e 7 M/s. To determine whether this conversion factor calculation is valid, we also analyzed the kinetics by utilizing the measurement of [02] with time shown in Figure 4a. These data are actually a sum of two effects: (1) the decrease in dissolved [O,] due to consumption by reaction (NADH can be autooxidized, i.e., oxidized without the HRP catalyst, but the autoxidation is negligible at the [NADH] used hereI3) and (2) the increase in [02] due to the leakage of air into the cell (which is a small but not insignificant effect). The latter change in [O,] can be monitored separately by running an identical experiment but leaving out the NADH and purging the solution of 0 2 prior to injection into thecell; the resulting [02] measurements are shown
1.8 X 7 . 5 5.6X 61.6X
10-7 10-7 ~
lP7 lP7
in Figure 4c. The slopes of Figure 4a and 4c must thus be subtracted: -9.0 X 10-8 M/s (slope of Figure 4a, a negatiue value) - 3.6 X 10-8 M/s (slope of Figure 4c, a positive value), which yields -1.2 X le7M/s; thus, the rate of decrease of [02] due to reaction is 1.2 X le7M/s. This latter rate is the same order of magnitudeas that found from the potential measurements. Both are also in reasonable agreement with a separate rate measurement made by following the decrease in absorbance at 340 nm due to NADH in a solution (identical to those used in the transport cell) bathing a piece of the immobilized enzyme membrane. All of these results are grouped together in Table I along with values from the literature12J3for free enzyme. (No prior kinetic measurements of this reaction using immobilized HRP have been reported.) Our measurements indicate an approximate 10-fold decrease in reaction rate for the immobilized enzyme as compared to the rates for free enzyme measured by other groups. The rate enhancer 2,4-dichlorophenolwas present in our experiments as well, which can increase the rate by a factor of 11-12 for free enzyme (see last line of Table I); it is possible that such an effect exists as well as for the immobilized case studied here. If so, the rate of reaction without 2,4-dichlorophenol would have been the same order of magnitude as the rate of autoxidation and, hence, unmeasurable; thus, we did not attempt to study this reaction in the absence of 2,4-dichlorophenol. Methylene blue is also known to enhance the rate of oxidation of NADH in the absence of the enzyme;14its main effect in the oscillatory system seems to be to stabilize the oscillations,although its exact role is still unknown. Evidence for damped oscillatory behavior was also observed in this system. Figure 5 shows a few cycles of a sawtooth-shaped oscillation in [02] observed in an experiment very similar to those described above. The main difference in conditions between this experiment and the kinetics studies was that the buffer solution was purged of some of the dissolved oxygen by bubbling dry N2 through the solutions; this decreased the [02] to approximately 50 pM, at which point oscillations begin. This oscillation is quite similar in shape and frequency to those which have been observed numerous times in experiments with the free enzyme. The amplitude and average [02] level are much larger, however; typical [O,] oscillations seen with free enzyme occur over the range 1-2 pM while those seen here vary over 10-50 pM. This observation of damped oscillations supports the possibility that sustained oscillations might also exist in the immobilized HRP system and, hence, in vivo where HRP is found immobilized in the cell wall.
Kinetics of the Peroxidase-Oxidase Reaction We continue our search for conditions under which the damped oscillation might become a sustained oscillation.
Discussion It is difficult to know with certainty whether the conditions of an in vitro experiment are close enough to in vivo conditions to be considered "the same" as in vivo. In studies of the PO reaction, several discrepancies between the two sets of conditions exist; among these discrepancies has been the physical environment of the enzyme. The results described hereshow that immobilization of the enzyme in an inert matrix does not adversely affect its activity and may even continue to allow nonlinear behavior, such as the damped oscillations observed here. Other differences between the in vitro conditions and probable in vivo conditions remain. Among these differences are the following: (1) The reactions in vivo likely occur in the dark; any photochemical effects which might occur in vitro would, thus, not be occurring in vivo. (2) Added modifiers such as 2,4-dichlorophenol and methylene blue are present in vitro; on the other hand, some species which are present in vivo may play the same role as these modifiers do in vitro. We also considered the question of whether the membrane potential could be used as an external probe of the rate of the reaction as it occurs inside the immobilization matrix. A series of papers by Friboulet and Thomas" explored the relationship between the potential across a BSA membrane containing immobilized acetylcholinesterase enzyme and factors such as the pH, external salt concentration, substrate concentration, etc. In addition, these authors reported observing oscillations in the potential which they attributed to an autocatalytic reaction coupled with the negative feedback due to slow substrate diffusion through the BSA matrix. Although their reports of oscillations in the acetylcholinesterase system have never been confirmed by others (and we have been unable to reproduce these experiments in our lab), the thorough study of the dependence of the membrane potential in such a system has some application to our work. In the Friboulet-Thomas system, the immobilization matrix is identical to that used here; Le., it consists of cross-linked BSA protein containing fixed charges. The magnitude and sign of these charges are pH-dependent and a function of the isoelectric point of the protein (approximately 5 for BSA). Hence, at pH values greater than 5 the BSA matrix is negatively charged while at pH values less than 5 it is positive. Measurements of the membrane potential under various conditions were carried out by Friboulet and Thomas, and it was found that at pH > 5 the BSA membrane is a cation exchanger, Le., cations will diffuse into the BSA membrane but anions will be somewhat rejected. Thus, the membrane potential in this system is a Donnan potential due to ion exclusion. The substrate in the experiment in which Friboulet-Thomas observed oscillationsis acetylcholinechloride; i.e., the relevant species is a cation. In addition, the FribouletThomas experiments were carried out at pH 7.5, so this substrate could freely enter the membrane under the reaction conditions. The reaction produces H+ as one of the products which would lead to a local decrease of the pH, rendering the BSA matrix less highly charged as the reaction proceeded. Since the acetylcholinesterase enzyme is known to exhibit a pH-dependent activity, the local decrease in pH was confirmed by noting a shift in the optimal pH toward more basic values for the immobilized case. In contrast, the substrate of the HRP-catalyzed reaction is NADH, which is actually a dianion. The reaction is carried out at pH 5; hence, the BSA matrix would be expected to be approximately neutral in charge and to, therefore, not reject the NADH anions. The values measured by Friboulet and Thomas
The Journal of Physical Chemistry, Vol. 97, No. 35,1993 9063 for a BSA-only membrane at pH 5 are comparable to those measured in our experiment. The magnitude of the potentials observed here are higher in the presence of HRP. This can be attributed to the Donnan effect as well, since H+ is a reactant in the PO reaction. Therefore, as the reaction proceeds, the BSA matrix becomes more negatively charged, thus rejecting more NADH anions and decreasing the potential, as observed (see Figure 4b).
Conclusion Biological oscillations occur at all levels in living systems, ranging from oscillatory membrane potentials in excitable cells to biorhythmsaffecting theentire organism. The chemical origins of these oscillationsmay be quite diverse, but the kinetic properties of enzyme-catalyzed reactions make them good candidates to be involved in the origins of biological oscillations. Horseradish peroxidase is found immobilized in the cell wall in vivo. We have determined that immobilization does not adversely affect theactivityoftheenzymeand that thePoreaction catalyzed by immobilized HRP displays evidence of nonlinear behavior. Thus, if the other differences between in vivo and in vitro conditions turn out to be unimportant, we will be able to conclude that oscillatory behavior is possible in vivo for the PO reaction. Whether such oscillatory behavior is, in turn, the basis for any biorhythms occurring in horseradish or other organism in which the PO reaction occurs would still be an open question. It is quite likely that the oscillations observed in this system occur in conjunction with spatiotemporal wave behavior, such as that which has been observed in the Belousov-Zhabotinskii reaction with immobilized catalyst.15 No direct observations of such waves were noted in these experiments, but this may be due to the fact that most of the important species in this reaction are transparent to visible light. We are exploring ways of visualizing spatiotemporal waves in this system if they exist.
Acknowledgment. We acknowledge support of this work by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and by the National Science Foundation under Grant CHE-8913895. References and Notes (1) Lewis,N.G.,Paice,M.G.,Eds. PlantCell~allPolymers,Biogenesis and Biodegradation; American Chemical Society: Washington, DC, 1989. (2) Yamazaki, I.; Yokota, K.; Nakajima, R. Biochim. Biophys. Res. Commun. 1965,21, 582. (3) Nakamura, S.;Yokota, K.; Yamazaki, I. Nature 1969, 222, 794. (4) Olsen, L. F.; Degn, H. Biochim. Biophys. Acra 1978, 523, 321. (5) Olsen, L. F.; Degn, H. Nature 1977, 267, 177. (6) Geest, T.; Steinmetz, C. G.; Larter, R.; Olsen, L. F. J. Phys. Chem. 1992, 96, 5678. (7) (a) Larter, R.; Olsen, L. F.; Steinmctz, C. G.; Geest, T. In Chaos in Chemical and Biochemical Systems; Field, R. J., Gydrgyi, L., Eds.; World ScientificPress: Singapore, 1993; pp 175-224. (b) Alexandre, S.;Dunford. H. B. Biophys. Chem. 1991,40,189. (c) Rys, P.; Wang, J. Biochem.Biophys. Res. Commun. 1992,186,612. (d) Samples, M. J.; Hung, Y.-F.; Ross, J. J . Phys. Chem. 1992, 96, 7338. (e) Baier, G.; Urban, P.; Wegmann, K. Z . Nafurforsch 1988, 43A, 995. (0 Olson, D. L.; Scheeline, A. Anal. Chim. Acra 1990, 237, 381. (8) Gross, G. G.; Janse, C.; Elstner, E. F. PIanta 1977, 136, 271. (9) Goldstein, L. In Methods of Enzymology; Academic Press: New York, 1976; Vol. 44, p 397. (10) Thomas, D.; Broun, G. In Methods of Enzymology; Academic Press: New York, 1976; Vol. 44, p 901. (11) Friboulet, A.; Thomas, D. Biophys. Chem. 1982,16, 139, 145, 153. (12) Avigliano, A.; Carelli, V.; Casini, A.; Finazzi-Agro, A.; Liberatore, F. Biochem. J. 1985,226. 391. (13) Halliwell, B. PIanta 1978, 140, 81. (14) Sevck, P.;Dunford. H. B. J . Phys. Chem. 1991, 95, 2411. (15) Winston,D.;Arora,M.;Maselko,J.;GbspBr,V.;Showalter,K.Narure 1991, 351, 132.
