Effects of Tyramine and 4-Aminophenol on the Oscillating Peroxidase

Article pubs.acs.org/JPCB

Effects of Tyramine and 4‑Aminophenol on the Oscillating Peroxidase−Oxidase Reaction Andrew G. McDonald* and Keith F. Tipton* School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland ABSTRACT: The peroxidase−oxidase oscillator, a model of biological oscillations, is usually studied in conjunction with the effector molecule, 2,4-dichlorophenol. In this account, we present evidence of the effects of a naturally occurring phenol, tyramine, on the reaction, and also those of the structurally similar 4-aminophenol. Whereas 2,4-dichlorophenol gives rise to sustained oscillations at 40 μM, it was discovered that tyramine promotes damped oscillations at a concentration of 120 μM. Oxidation of NADH was completely inhibited by 4aminophenol and ascorbate. In separate experiments, the peroxidase-catalyzed ring coupling of tyramine and 4-aminophenol was observed, which in the case of tyramine, may provide an explanation for the damping of oscillations.

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species and are called “compounds” in consequence; for example, Per6+ (compound III) is formed by the combination of Per3+ (ferriperoxidase) with superoxide radical. Most studies on this system have included micromolar or submicromolar concentrations of at least one cofactor, most usually 2,4-dichlorophenol (2,4-DCP), which has been reported to enhance the oscillations by catalyzing the production of free radical reaction intermediates, such as NAD radical (NAD•).14 The presence of methylene blue (MB), which is thought to assist in the breakdown of the Per6+,15 in addition to DCP, has been variously claimed to increase the lifetimes and amplitudes of oscillations. The concentrations of these electron acceptors may act as parameters of the dynamic behavior of the reaction. By varying the concentration of 2,4-DCP, Geest et al. were able to demonstrate a period-doubling route to chaos.11 Studies by Hauser and Olsen16 into the actions of a variety of naturally occurring phenols on this oscillator suggested a possible mechanism of action. Denoting the phenolic compound by XOH, and NADH and NAD• by AH and A•, respectively, the following partial reaction scheme14,16 has been used to describe the postulated mode of action of the phenol in the PO reaction

INTRODUCTION Oscillating behavior is known to occur widely in biochemical systems.1 Rhythms generated at the level of gene expression are well-established, 2 but they also occur at the level of metabolism.3,4 The nonlinear kinetics of enzymes in a metabolic pathway, coupled with feedback regulation, is a necessary, if not sufficient condition for concentration oscillations to appear. The oscillating peroxidase−oxidase (PO) reaction, one of the few single-enzyme oscillators that has been characterized to date,5,6 is the aerobic oxidation of NADH by oxygen in the presence of haem-peroxidases, which may be represented by the overall reaction 2NADH + O2 + 2H+ → 2NAD+ + 2H 2O

(1)

The first observations of the nonlinear kinetics evinced by the reaction were of damped oscillations when a system of peroxidase from horseradish and NADH was held open to oxygen.7 Subsequent work by several other research groups has revealed that sustained, quasiperiodic, and chaotic oscillations in the concentrations of dissolved oxygen, NADH, and various intermediate forms of the enzyme are possible when the reaction system is held open to both NADH and oxygen.8−10 This oscillator is the only known single-enzyme biochemical reaction to exhibit chaos, which makes it useful as a laboratory model of nonlinear dynamics in biological systems.11 The earlier literature on this oscillator has been comprehensively reviewed by Scheeline et al.12 Peroxidases from several species, including the horseradish, can participate in one-electron transfer reactions, which alter the oxidation state of the haem moiety. Five such oxidation states of the haem iron center are known to be involved in the peroxidase reaction, which may be denoted by Per2+ through Per6+, Per3+ being the native ferriperoxidase.13 Some of the oxidation states are complexes of free enzyme with other © 2013 American Chemical Society

XOH + Y • → XO• + YH

(2)

XO• + AH → XOH + A•

(3)

This serves to catalyze the production of NAD• (A•) from an electron acceptor Y•, which may be one of several components of the reaction, including superoxide and Per6+.14,17 Hauser and Olsen16 have proposed that the oxidation half-wave potential (E1/2) of the phenoxyl radical determines its effectiveness in augmenting the PO reaction and that the higher that this value Received: July 8, 2013 Revised: November 15, 2013 Published: December 10, 2013 18

dx.doi.org/10.1021/jp406707s | J. Phys. Chem. B 2014, 118, 18−25

The Journal of Physical Chemistry B

Article

the re-equilibration of oxygen were used to provide a measure of the oxygen mass-transport constant k−t19 from the equation

is, the more likely it is that the compound will produce more complex patterns of oscillations. The present report focuses on the effects of two phenols not previously studied with the PO reaction, the naturally occurring tyramine (4-[2-aminoethyl]phenol) and its structural analogue, 4-aminophenol. Oxidative ring coupling of tyramine and 4aminophenol was also observed. This provides further evidence for the involvement of a free radical form of the phenol in the oscillatory PO reaction. The involvement of radical species was investigated by the use of ascorbate and superoxide dismutase (SOD; EC 1.15.1.1).

[O2 ] = [O2 ]eq (1 − e−k−tt )

(4)

where t is the time elapsed from the introduction of the 2% oxygen gas stream, [O2] the measured oxygen concentration of the solution, and [O2]eq its concentration at equilibrium. Once the dissolved oxygen concentration had reached a maximum, the NADH line was placed approximately 2 mm beneath the surface of the solution and infusion of the 80 mM stock NADH started at between 1.4 and 1.6 mM/h. Shortly after oscillations commenced, the influx rate of NADH was decreased to between 1.0 and 1.2 mM/h in order to maintain a constant average concentration in the reaction medium. For oxidative ring coupling experiments catalyzed by horseradish peroxidase, tyramine and 4-aminophenol in the concentration range of 0−300 μM were separately dissolved in 0.1 M sodium acetate buffer at pH 5.1, along with 300 μM hydrogen peroxide, and incubated at 25 °C in the spectrophotometer. Horseradish peroxidase, at a final concentration in the range of 0.08−0.16 μg/mL, was added, and thereafter, a series of absorbance spectra were recorded at 5 min intervals for up to 45 min.

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MATERIALS AND METHODS Horseradish peroxidase (HRP; EC 1.11.1.7; immunoassay purity, containing approximately 300 pyrogallol units per mg solid), 4-[2-aminoethyl]phenol, L-ascorbic acid, 2,4-dichlorophenol (2,4-DCP), hydrogen peroxide (30%), and MB were obtained from Sigma (Poole, Dorset, U.K.). NADH was purchased from Sigma as the disodium salt. 4-Aminophenol was purchased from Aldrich. The computer-controlled system and general procedures used for these studies were essentially as described previously.18 A 2 mL solution of 40 U/mL HRP, 20 μM 2,4-dichlorophenol, and 0.2 μM MB in 0.1 M sodium acetate buffer, pH 5.1, were equilibrated in a 1 cm quartz cuvette to 25 °C in a UV/vis spectrophotometer (Kontron Uvikon 901). An oxygen/nitrogen gas stream of final composition 2% in oxygen was bubbled at 20 mL/min into the solution, the end of the gas line being placed just beneath the meniscus of the liquid (bubbling did not impinge upon the light path of the spectrophotometer). Gas flow was regulated by two mass flow controller devices (Sierra Instruments Model 902c). NADH was infused into the solution using a stepper-motor syringe infusion pump from an 80 mM stock in a 100 μL Hamilton syringe fitted with a custom needle of length 40 cm and with an internal diameter of 0.11 mm. The mixture was continuously stirred from beneath by a magnetic stirrer (Rank Bros, U.K.); the stirrer speed was 1160 rpm, for which the mixing time was estimated to be approximately 2 s.18 Mixing time was determined by monitoring the absorbance of a dye after injection into a stirred aqueous solution and calculated as the time taken to reach a fixed percentage of the final equilibrium position. The dissolved oxygen concentration was measured using a Microelectrodes dioxygen probe and meter (Models MI-730 and OM-4, Microelectrodes, New Hampshire, U.S.A.). A Teflon lid, with holes for the reagent lines, oxygen probe, and gas efflux, was affixed to the cuvette and sealed to provide a gas headspace of constant oxygen composition immediately above the liquid. Data from the spectrophotometer and oxygen meter were sampled using a 12-bit data acquisition card (PCI1200, National Instruments, TX, U.S.A.). The spectrophotometer and syringe pump were controlled throughout experiments by means of a LabVIEW (National Instruments) program written in-house. Absorbance data were acquired every 6 s, and dissolved oxygen data were sampled once per second. The absorbances of NADH and compound III were followed at 360 and 418 nm, respectively. At the start of an experiment, the solution was purged of oxygen by bubbling a pure nitrogen stream into the solution of enzyme and the electron acceptors 2,4-DCP and MB, the acquisition of oxygen data commenced, and after oxygen concentrations had reached a minimum, gaseous oxygen was introduced with a composition of 2%. The data obtained from

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RESULTS AND DISCUSSION A typical set of data obtained is shown in Figure 1 and provides a standard to which the results of replacing 2,4-dichlorophenol

Figure 1. Sustained oscillations in dissolved oxygen and peroxidase compound III, measured at 418 nm. The reaction mixture was comprised of 40 U/mL HRP, 20 μM 2,4-dichlorophenol, and 0.2 μM MB in 0.1 M sodium acetate buffer at pH 5.1. The final NADH influx rate was 1.2 mM/h. Other details are given in the text.

by tyramine and 4-aminophenol may be compared. The oscillation character with 2,4-DCP was reproducible and of a single period (period one). The experiments with the aminophenols proceeded along two lines, the first of which involved the substitution, at varying concentrations, of the aminophenol for 2,4-dichlorophenol. The second study was a spectral analysis of the formation of ring-coupled phenolic species. In a series of experiments, the concentration of tyramine was varied in the range of 0−120 μM in the absence of 2,4dichlorophenol. Three representative data sets are shown in 19



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transport constant k−t (eq 4) was 1.1 ± 0.1 × 10−2 s−1. Damped oscillations were obtained in all cases, with the number of oscillations increasing as the initial concentration of tyramine in the reaction medium was raised. Tyramine added to a working standard oscillator, such as that shown in Figure 1, at a final concentration of 25 μM, had no effect on the pattern of oscillations obtained (data not shown). In the absence of tyramine, the oxidation of NADH proceeded nonlinearly, reaching a new steady-state value at lower oxygen concentration in under 5 min. At the highest concentration of tyramine, 24 oscillations were obtained before the system settled to a nonoscillatory steady state. Oscillations were also observed in NADH concentration, when measured at 360 nm, which followed an identical pattern to those in dissolved oxygen. 4-Aminophenol was found to be a potent inhibitor of the oscillatory PO reaction. When 4-aminophenol was added before or during the course of the reaction, oscillations did not commence in the former instance and immediately ceased in the latter. In the experiment in which the data in Figure 3a were obtained, 4-aminophenol replaced 2,4-DCP at a concentration of 25 μM; no oscillations were observed. As a control, the experiment was repeated using 2,4-DCP and otherwise identical reagent concentrations, and sustained oscillations appeared. When 4-aminophenol, at a final concentration of 25 μM, was added to the reaction mixture during the course of an oscillatory run, the result was total cessation of oscillations and the return of oxygen levels to equilibrium with the gas

Figure 2. Damped oscillations in the presence of increasing levels of tyramine. The reaction mixture was comprised of 40 U/mL HRP, 0.2 μM MB, and tyramine dissolved in 0.1 M sodium acetate buffer, pH 5.1. The final NADH influx rate was 1.2 mM/h. Tyramine concentrations were (a) 0, (b) 60, and (c) 120 μM.

Figure 2, for the values 0, 60, and 120 μM. All other conditions were those of the standard set (Figure 1), except that the final NADH influx rate varied between experiments owing to the need to maintain a constant average concentration in the cuvette. The mean value of the computed oxygen mass-

Figure 3. The effects on the PO reaction of (a) 25 μM 4-aminophenol in the absence of 2,4-DCP; (b) a pulse of 4-aminophenol at a final concentration of 25 μM on the oscillating PO reaction, in the presence of 20 μM 2,4-dichlorophenol. The other conditions were those of Figure 1. In (a), after purging of the solution with pure nitrogen and introduction of 2% oxygen after 1000 s, no change in the steady concentration of oxygen was observed after NADH infusion commenced, at 1.6 mM/h, although acid-catalyzed autoxidation of NADH was observed after its infusion had stopped. 20



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duration of the experiment, in keeping with the results of Valoti et al.21 No reaction occurred in the absence of enzyme (not shown). Some phenols, such as 2,4-DCP, are known to sustain oscillations in the PO reaction,14,16 and tyramine will presumably pass through a free radical intermediate through the action of the enzyme. The damping of oscillations may be explained by the irreversible conversion of tyramine to dityramine, which removes the active phenol. The hydrogen peroxide required for this reaction will be formed by the autoxidation of NADH in the reaction22

phase, as shown in Figure 3b. Comparing the data in Figure 3a with those of Figure 2a, it is evident that little or no decline in the steady-state oxygen concentration has taken place in the presence of 4-aminophenol. It is known that peroxidases from some sources can initiate oxidative ring coupling of tyrosine and tyramine in the presence of H2O2,20,21 and a mechanism for this was proposed by Gross and Sizer,20 as shown for the case of tyramine in Figure 4. A set

NADH + H+ + O2 → NAD+ + H 2O2

Oxidative ring coupling with 4-aminophenol was also observed, although the rate of the reaction was considerably faster under similar conditions and reagent concentrations. In addition, the reaction progressed rapidly at lower concentrations and even in the absence of enzyme, in contrast with the case of tyramine. In the presence of 50 μM 4-aminophenol, 300 μM H2O2, and 0.08 μg/mL HRP, the reaction was essentially complete within 5 min of addition of the enzyme. When enzyme was omitted, the reaction proceeded more slowly but with the same yield over 45 min as that in the first case. The reaction was observed both in the presence and the absence of the enzyme but not in the absence H2O2, indicating that the first step in the oxidation process requires hydrogen peroxide. The data are displayed in Figure 6. As might be expected in view of the involvement of radical species in this process, the addition of 100 μM ascorbate before HRP suppressed the ring coupling (data not shown). To provide a quantitative measure of the rates of product formation, a simple exponential model was fitted to the data sets shown in Figure 5 at 290 nm. The model used was of the form x = a + be−ct for which the values of the parameters a and b and the apparent first-order rate constant c were found by nonlinear regression to be a = 0.18, b = −0.01, and c = 0.05 min−1, with χ2 = 3.1 × 10−7. Neither a first-order nor a secondorder kinetic model fitted the spectral data for the formation of di(4-aminophenol) shown in Figure 6, although a generalized double-exponential function did. A simple dimensionless model of the oscillating PO reaction has been developed by Watanabe and Inaba.14 This was modified to incorporate a decay function for the phenol, with the apparent first-order parameter determined for dityramine formation, as shown in the Appendix. In this model, the rate constant for the formation of NAD• from XO•, k7, is dependent upon the concentration of the phenolic compound. Numerical integration of the model in XPPAUT (http://www.math.pitt. edu/~bard/xpp/xpp.html), using a fourth-order Runge−Kutta method, reproduced the damped oscillations in oxygen (variable B) observed with tyramine when the parameter k7 was varied, as shown in Figure 7a−c. Using XPPAUT, a bifurcation analysis of the model, shown in Figure 7d, revealed the existence of an unstable periodic orbit between the onset of stable periodic oscillations at k7 ≈ 19.3 and a Hopf bifurcation at k7 ≈ 69.4. Between these two values, initial conditions outside of the unstable periodic orbit were attracted to the stable limit cycle surrounding it, whereas simulations started closer to the steady state were damped. Although our simulations were performed with a lower value of k5 than that of the original publication, the original value of k5 produces a similar figure, with the Hopf bifurcation appearing at a higher value of k7. The existence of such complex behavior was not mentioned in the original treatment of this system.14

Figure 4. Mechanism of formation of dityramine from tyramine by the action of peroxidase.20

of experiments was performed to verify if this reaction would take place with horseradish peroxidase, under the conditions used in this work. Figure 5 shows absorbance spectra of a solution containing 100 μM tyramine, 300 μM hydrogen peroxide, and 1.6 μg/mL peroxidase in 0.1 M sodium acetate buffer at pH 5.1. The reaction was started by the addition of enzyme. In the range studied, 240−320 nm, the absorbances at all wavelengths were found to increase throughout the 45 min

Figure 5. Spectral changes accompanying conversion of tyramine to dityramine by the mechanism of Figure 4. Horseradish peroxidase, with a final concentration of 1.6 μg/mL, was added to a solution of 100 μM tyramine and 300 μM hydrogen peroxide in 0.1 M sodium acetate at pH 5.1. The temperature was held at 25 °C. The absorbance spectra recorded at 5 min intervals following the introduction of the enzyme show an increase in absorbance at all wavelengths between the times indicated on the graph. 21



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Figure 6. Oxidative ring coupling of 4-aminophenol. All reagents were dissolved in 0.1 M sodium acetate buffer at pH 5.1. Other conditions were those of Figure 5. Absorbance spectra were taken at 5 min intervals after the addition of the enzyme. In (a−d), curves marked A and B were obtained at t = 0 and 45 min, respectively. In the presence of HRP, at the highest concentration of 4-aminophenol, the product was already forming during the scan taken at time zero; (a) 25 μM 4-aminophenol, 300 μM H2O2, 0.08 μg/mL HRP; (b) 50 μM 4-aminophenol, 300 μM H2O2, 0.08 μg/mL HRP; (c) 50 μM 4-aminophenol, 300 μM H2O2, HRP omitted; (d) 25 μM 4-aminophenol, H2O2 and HRP omitted.

form the dimer. The heuristic model that we have employed can reproduce the increase in the quality of damped oscillations observed in the experiments with tyramine but is deficient in other respects, most notably in that the phenolic is presumed to interact only with nonenzyme species. A more comprehensive model, which may account for both aspects of the reaction of phenolic, has been proposed by Bronnikova et al.26,27 As might be expected in view of the involvement of several radical species in this process, the addition of 100 μM ascorbate before HRP completely suppressed the ring coupling (data not shown). The addition of ascorbate at a final concentration of 10 mM during the progress of an oscillating reaction caused complete inhibition of NADH oxidation, with the dissolved oxygen re-equilibrating with the gas phase. The reaction was also inhibited following the addition of SOD (30 U), presumably as a result of prevention of Per6+ formation. Ascorbate has been used in studies of the peroxidase reaction to minimize the levels of substrate-derived radical species,28 but its effects on the PO oscillating system have not, to our knowledge, been reported before. 4-Aminophenol is known to be a good substrate for peroxidase,23,29 but unlike tyramine, it was found to be a potent inhibitor of the oscillatory PO reaction. Other

The rate of oxidation of phenolic compounds to phenoxyl radicals by peroxidase has been shown to be dependent on the redox potential of the phenol, which governs the rate of electron transfer to the enzyme, and the affinity for binding appropriately to the enzyme active site.23,24 Hauser and Olsen16 argued that the phenolic half-wave potential was also an important determinant of the ability of such compounds to promote oscillations in this system and that, in particular, phenols whose half-wave potentials fall in the range of approximately 780−880 mV are able to induce sustained oscillations. The half-wave potential of tyramine, determined by Monzani and co-workers to be 830 mV (pH 5.0),25 lies within this range. This would be consistent with it being a promotor of sustained oscillations; however, the consumption of tyramine will result in a decrease in this activity over time owing to its removal from the reaction as dityramine. Our observation that tyramine was capable of sustaining oscillations for longer periods as its concentration was increased is consistent with the damping of the oscillations resulting from tyramine consumption. The formation of dityramine is known to pass through a free radical intermediate, which may be involved in competing processes linked to NADH and peroxidase, with oscillations ceasing when the tyramine radicals are removed to 22



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Figure 7. Numerical simulations (a−c) and bifurcation diagram (d) of the Watanabe−Inaba mathematical model of the PO reaction.14 Dissolved oxygen concentration (B) and time units are dimensionless. For the simulations in (a−c), k = 1 × 10−3, while for the bifurcation analysis in (d), k = 0, so that ϕ(t) = 1. The number of oscillations appearing during damping to a steady state increases with k7: k7 = (a) 5, (b) 10, and (c) 15. The bifurcation diagram (d) demonstrates the coexistence of a stable steady state with a periodic orbit in B−k7 space. Other parameters were k1 = 0.07, k2 = 0.0032, k3 = 20.0, k4 = 2.0, k5 = 2000.0, k6 = 300.0, B0 = 1.0, and Z0 = 0.01. Key: SS = steady state; BP = branch point; HB = Hopf bifurcation. See the text for further details. 23



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studies30,31 have used ESR spectroscopy to show the existence of 4-aminophenol radicals in the peroxidase-catalyzed reaction, but their effects on the oscillatory oxidation of NADH by HRP has not been examined before. The half-wave potential of 4aminophenol has been reported to be considerably lower than that of tyramine, at 43 mV,32 which would suggest its inability to support oscillations, according to the hypothesis of Hauser and Olsen,16 and as observed in the present results. The spectral evidence was consistent with facile oxidative ring coupling in the presence of hydrogen peroxide, a process that is known to be catalyzed by horseradish peroxidase.29 Although there is evidence that phenoxyl radicals, which are intermediates in such reactions, can inactivate peroxidase at millimolar concentrations of H2O2 and phenol,33 such inhibition would not be expected to occur at the concentrations used in the present work. In accord with this is the report that 4-aminophenol does not inhibit plant peroxidase, although it is a suicide inhibitor of ascorbate peroxidase (EC 1.11.1.11).31 It is possible that 4-aminophenol is acting to terminate the radical process by acting as a two-electron acceptor with the formation of the corresponding quinone.23 This and the possible interaction of 4-aminophenol with NADH, as has been shown for the phenol scopoletin,34 will require further investigation. It has been suggested that the oscillatory behavior may be important in vivo in protecting peroxidase from inactivation.35 Because tyramine is a naturally occurring compound, it could be an effector of the oscillatory behavior, but the variety and complexity of phenol derivatives in plants makes it difficult to predict the overall responses without comprehensive studies on the effects of such phenolic mixtures on this system. Furthermore, the suppression of oscillations by ascorbate and SOD suggests that they may not occur under in vivo conditions.

Scheme 1. Watanabe−Inaba Model Reaction Scheme,14 Modified to Include Dimerization of the Phenoxyl Radical, XO•, According to 2 XO• → (XO)2a

a

The concentrations of species in boldface are variables of the model.

dB = k1(B0 − B) − k 3BX dt

(5)

dX = k 2 + k 7ϕ(t )Y 2 − k 3BX − k6XZ dt

(6)

(2) NAD+ is assumed not to have any effect upon the reaction.36 (3) The NAD• intermediate is assumed to form autonomously according to zero-order kinetics.36 (4) The dismutation reaction (k4) is believed to be 2O•− 2 + 2H+ → O2 + H2O2, the products of which are assumed not to contribute significantly to the reaction. To test this, we have confirmed that the addition of +k4Y2 to dB/dt does not alter significantly the behavior of the model shown in Figure 7d but only the positions of bifurcation points on the k7 axis and the exchange of the branch point (BP) with a limit point (fold). (5) The “feedback” term [NADH][XO•] was assumed by the original authors to be quadratic with respect to the concentration of superoxide. Thus, both XO• and XOH are implicit to the model. The function ϕ(t), which is absent from the original, enables it to be converted to a nonautonomous system according to ϕ(t) = e−kt, where k is an apparent first-order rate constant for the formation of dimer (XO)2.

dY = k 3BX − k4Y 2 − k5(Z0 − Z)Y dt

(7)

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Corresponding Authors

dZ = k5(Z0 − Z)Y − k6XZ dt

(8)

Notes

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APPENDIX: MODIFIED WATANABE−INABA MODEL The equations of the mathematical model described by Watanabe and Inaba14 take the form

AUTHOR INFORMATION

*E-mail: [email protected]. *E-mail: [email protected]. The authors declare no competing financial interest.

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where t is dimensionless time and the dependent variables B, X, Y, and Z correspond to dimensionless concentrations of dissolved oxygen, NAD•, superoxide, and oxyperoxidase (Per6+). The concentration of native (ferri-)peroxidase will be given by Z0 − Z. Scheme 1 shows the reaction network on which it is based. All modeled species are in aqueous phase, with the exception of the constant B0, which represents gaseous oxygen. The model is phenomenological in nature, and several assumptions were made:14 (1) The pH and the average NADH concentration remain constant.

ACKNOWLEDGMENTS Financial support for this work by Forbairt, Trinity College Dublin, and BioResearch Ireland is gratefully acknowledged. REFERENCES

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Effects of Tyramine and 4-Aminophenol on the Oscillating Peroxidase

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