J. Phys. Chem. 1995, 99, 1974-1979
1974
New Experimental Methods toward the Deduction of the Mechanism of the Oscillatory Peroxidase- Oxidase Reaction Yu-Fen Hung and John Ross* Department of Chemistry, Stanford UniversiQ, Stanford, California 94305 Received: September I , 1994; In Final Form: November 23, 1994@
We study the peroxidase-oxidase reaction with new experimental methods useful for the deduction of oscillatory mechanisms. We measure four species, oxygen, reduced nicotinamide adenine dinucleotide (NADH), native horseradish peroxidase (HRP), and compound I11 (coIII), simultaneously to determine their roles in the reaction mechanism. We measure the relative amplitude and phase shift relations of these species and perform concentration shift regulation and destabilization, qualitative pulsed-species response, and quench experiments. Within a scheme of categorization of oscillatory reaction mechanisms, we identify oxygen as a type Y essential species and COIIIa type Z essential species. Both NADH and the native HRP enzyme are nonessential species, likely of type C. With the results of the experiments presented combined with other known facts about the experimental system, the category of the oscillator is determined to be 1CW. The assignment of the roles of the species and the categorization of the oscillator allows for the deduction of parts of the reaction mechanism.
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Introduction
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The peroxidase-oxidase (PO) reaction, widely studied, exhibits a variety of different types of nonlinear dynamics. The overall reaction is 2NADH
+ 0, + 2H'
2NAD' 4-2H,O
where NADH is reduced nicotinamide adenine dinucleotide, and HRP is the horseradish peroxidase enzyme. Complex dynamics such as limit cycle oscillations,' chaos,2bistability between two stationary state^,^ quasiperiodicity? and mixed-mode oscillat i o n ~have ~ all been observed experimentally. Many different models, both abstract6-* and detailed?-l2 have also been proposed for this oscillatory reaction. There are proposed systematic methods of studying oscillatory dynamics by assigning roles to the different species in the system, indicative of the connectivity of essential species in the reaction mechanism, and categorizing the oscillator^.'^^'^ The development of these methods was based on model calculations, but they have also been applied recently to experimental result^'^,'^ and found to be effective, even when the experimental measurements are limited due to the lack of appropriate measurement techniques. These methods and tests also allow for the deduction of some reaction steps in the system by directly measuring the connectivity of the different species in the reaction. In this study, we apply some of the tests suggested in refs 13 and 14 experimentally to the PO reaction to assign roles to four of the detectable species in the system: oxygen, NADH, the native enzyme (Per3+),and compound 111 (coIII). This information is then used to categorize the oscillator. These tests include comparing the relative amplitude and phase shifts of the different oscillatory waveforms of these compounds, concentration shift regulation and destabilization experiments, qualitative pulsed species-response tests, and quench experiments. These and other categorizationmethods are summarized in ref 17. We will give
* To whom correspondence should be addressed. @
Abstract published in Advance ACS Absrracfs, January 15, 1995
0022-365419512099-1974$09.0010
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a brief description of these tests and how they are used for the assignment of the species before presenting the experimental results. Experimental Section The experimental apparatus used to perform the PO reaction consists of a well-stirred reactor with the reactants oxygen and NADH supplied continuously; it is a modification of that described in ref 4. A schematic of the apparatus is shown in Figure 1. The reactor is an 18.5 x 20 x 37.5 mm quartz cuvette with two spacers (5 x 6 x 5 mm and 7 mm from the bottom of cuvette), used to decrease the light path from 20 to 8 mm in certain runs and also to serve as baffles to enhance mixing (custom-made by Hellma Cells, Inc., QS 402.018). The reactor 0 1995 American Chemical Society
The Oscillatory Peroxidase-Oxidase Reaction. 1 is set in a thermostated jacket that is situated in a spectrophotometer. The measuring devices for the four compounds studied consist of a photodiode-array ultraviolet-visible spectrophotometer (Beckman, DU-7500) and an oxygen electrode (Microelectrodes, Inc., Model MI-730). The NADH concentration is measured at 340 nm while native H W and coIII are measured at 403 and 418 nm, respectively. Spectrophotometric data are recorded every 3 or 4 s rather than more frequently due to a slight photochemical effect on the reaction.'* Oxygen is sampled once every second. Oxygen and NADH are flowed into the reactor which has an outflow for oxygen only. The products, mainly NAD+ and H20, in this reaction are considered to be inert for the time frame (usually < 3 h) of the experiments and do not feed back into the reaction. Furthermore, the volume change of the reacting solution during the experiments is negligible (usually < 1%). Oxygen diffuses into the solution through a gas headspace as a mixture of nitrogen and compressed air flows which are controlled by two mass flow controllers (Vacuum General, Inc., UltraFlo Model UC2-21) and premixed by a two gas-blender (Union Carbide, Linde Division, Model FM 4621-13); NADH is supplied by a syringe pump (Harvard Apparatus, Model 22). The gas humidifier moistens the dry gas before it enters the reactor and is necessary to prevent a decrease in the volume of the reacting solution due to evaporation. The solution is well mixed by a stirring motor and stainless steel shaft (Spectrocell, Inc., MTR-11D) fitted with a stirring blade (made of delrin) that stirs at 1000 f 50 rpm. Complete mixing occurs within a few seconds, evidenced by adding dye to the solution. The chart recorder is used for the initial calibration of the oxygen electrode at the start of every run and monitors the oxygen response during experiments. The two computers control the gas inputs and record the outputs of the oxygen electrode and the voltage signals of the gas inflows. The reactor is maintained at 28 "C for all experiments. Typical starting solutions for the experiments described here consist of the following: 0.1 M sodium acetate buffer at pH 5.1 (sodium acetate made by Mallinckrodt), 12-24 pM of oxygen depending on the initial flow rate (1.0-2.0%), 10-25 pM of 2,4-dichlorophenol (DCP, Sigma Chemical Co.), 0.1 pM of methylene blue (MB, Sigma), and 0.40-0.80 ,uM of horseradish peroxidase enzyme (HRP) purchased as a sodium phosphate suspension at pH 6.0 from Boehringer Mannheim, grade I with purity number greater than 3.0. NADH (0.15 M, Boehringer Mannheim, grade I disodium salt) solution is made with distilled, deionized water purged with nitrogen beforehand to eliminate the oxygen in the water and is inflowed at a rate of 15-40 p L h . The purging is necessary in order to minimize the auto-oxidation that occurs in the solution. The buffer (made with distilled, deionized water), HRP, and NADH are stored in the dark at 4 "C before use. DCP stock solution (0.01 M) is made by dissolving DCP in ethanol (Gold Shield Chemical Co., 200 proof) and is stored in the dark at 23 "C. MB solution (1 mM) is made by dissolving MB in distilled, deionized water and also stored in the dark at 23 "C. The reacting solution is 7.0 or 7.5 mL depending on the run. For an analysis of the experimental conditions and the variables used in the PO reaction, see ref 19.
Results The experiments described here are designed according to categorization tests formulated in refs 13 and 14. There are two types of species, essential and nonessential, and two main categories, 1 and 2, involved in oscillatory systems according to the analyses performed on 25 abstract and detailed models. The essential species, types X, Y, Z, and W, are necessary for
J. Phys. Chem., Vol. 99, No. 7, 1995 1975
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Figure 2. Measurements of the relative amplitudes of oxygen, NADH, native HRP, and coIII oscillations (from typical oscillatory reaction). Typical external constraints and initial conditions: 1.25% oxygen flow (2.0 m L / s total gas flow rate), 28.0 pL/h NADH flow (0.15 M solution), 0.65 pM of HRP, 10 pM of DCP, 0.1 p M of MB, and 7.5 mL of solution in reactor. The relative values are calculated according to the formula in the text. The relative amplitudes vary slightly depending on the changes in the absolute concentrations, Le., the larger the absolute amplitudes, the larger the relative amplitudes of the three species with respect to oxygen, but the general trend, oxygen > coIII > Per3+ > NADH does not change.
the oscillatory behavior in the system and must be considered in any mechanism describing the oscillator, whereas the nonessential species, types A, B, and C, are not necessary for oscillations but are reactants that produce the essential species, or inert products. See refs 13 and 14 for the definitions of the different types of essential and nonessential species. Category 1 oscillators are further separated into lB, lCX, and 1CW oscillators, where 1CX and 1CW are basically the same except for an additional essential species of type W in category 1CW. The different tests are mainly conducted close to a Hopf bifurcation, mostly a supercritical Hopf, on either the stationary or the oscillatory side of the bifurcation. The supercritical Hopf bifurcation region used in these experiments is located by varying the enzyme concentration for various oxygen and NADH inflows. With this experimental apparatus, stationary states typically occur at lower enzyme concentrations ( 2x -> Y
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Conclusions These experiments have allowed for a more in depth understanding of the PO reaction by revealing information about the identity and the importance of the species measured for oscillations. C a l c u l a t i ~ n s ' ~are ~ ' ~also ~ ' ~performed ~~~ with the abstract DOP model6 and the detailed models AI' and CI2 to assign roles to the species and to determine the oscillatory category of these two mechanisms for comparison to the experimental results. Depending on the parameter region chosen, the oscillatory category of these models varies. The DOP model has been determined to be a category 2 oscillat ~ r , ' ~while , ' ~ both models A and C belong in the 1CW category for certain sets of parameters.20 Therefore, these experiments suggest that the core mechanism considered in these two detailed models should be the same as in the experimental system. Figure 7 shows the prototypical 1CW oscillator and model A, with the autocatalytic cycle in bold lines and the relevant essential species in circles next to the compound, which categorizes it as a 1CW oscillator.20 We can see the similarity of the two networks as COIII(Z) reacts with NAD' (X) to form more of the radical and oxygen (Y) reacting with NAD' (X) to form 02.- (W). Hence, the experiments presented here are useful in the deduction of core mechanisms of oscillatory reactions by determining the roles of the species and the oscillatory category. The other reactions can be added as more experimental evidence becomes available for the determination of the complete model. Although the core mechanism of the oscillatory PO reaction is incorporated in models A" and C,'* evidenced by the experiments presented here, there are discrepancies, such as differences in the oscillatory waveforms and complex dynamics exhibited in these models compared to the experimental measurements.I8 A new mode1,'8s22which builds on model C and is also a category 1CW oscillator for the set of parameters considered, exhibits not only similar waveforms and phase shifts as the measured species but also the various complex dynamics that have been observed experimentally in the PO reaction.
7
10
Figure 7. Reactions and network diagram of the category 1CW oscillator and the network diagram of model A (redrawn from ref 21 with the numbers corresponding to Table I in ref 21). The bold lines in model A highlight the reactions that characterize the autocatalytic cycle, and the types of essential species are listed in circles next to the compounds.
Acknowledgment. This work was supported in part by the National Institutes of Health. We thank Dr. Lisa L. Skrumeda for her help with the quench experiments. We also thank Dr. Igor Schreiber for helpful discussions and comments. References and Notes (1) Nakamura, S.; Yokota, K.; Yamazaki, I. Nature 1969, 222, 794. (2) Olsen, L. F.; Degn, H. Nature 1977, 267, 177. (3) Degn, H. Nature 1968, 217, 1047. (4) Samples, M. S.; Hung, Y.-F.; Ross, J. J. Phys. Chem. 1992, 96, 7338. (5) Steinmetz, C. G.; Geest, T.; Larter, R. J. Phys. Chem. 1993, 97, 5649. (6) Olsen, L. F.; Degn, H. Biochem. Biophys. Acta 1978, 523, 321. (7) Olsen, L. F. Phys. Lett. 1983, 94A, 454. (8) Alexandre, S.; Dunford, B. H. Biophys. Chem. 1991, 40, 189. (9) Yokota, K.-N.; Yamazaki, I. Biochemistry 1977, 16, 1913. (10) Fed'kina, V. R.; Ataullakhanov, F. I.; Bronnikova, T. V. Biophys. Chem. 1984, 19, 259. (11) Aguda, B. D.; Clarke, B. L. J. Chem. Phys. 1987, 87, 3461. (12) Aguda, B. D.; Larter, R. J. Am. Chem. SOC.1991, 113, 7913. (13) Eiswirth, M.; Freund, A,; Ross, J. Adv. Chem. Phys. 1991,80, 127. (14) Chevalier, T.; Schreiber, I.; Ross, J. J. Phys. Chem. 1993, 97. 6776. (15) Skrumeda, L. L. Ph.D. Thesis, Stanford University, 1993. (16) Stemwedel, J. D. Ph.D. Thesis, Stanford University, 1993. (17) Stemwedel, J. D.; Schreiber, I.; Ross, J. Adv. Chem. Phys., in press. (18) Hung, Y.-F. Ph. D. Thesis, Stanford University, 1994. (19) Olson, D. L.; Scheeline, A. Anal. Chim. Acta 1993, 283, 703. (20) Schreiber, I.; Hung, Y.-F.; Ross, J., manuscript in preparation. (21) Aguda, B. D.; Larter, R. J. Am. Chem. SOC.1990, 112, 2167. (22) Hung, Y.-F.; Schreiber, I.; Ross, J. J. Phys. Chem., in press. JP942379F
