A biochemical oscillator : experimental and theoretical studies of the peroxidase-oxidase reaction

Abstract

The peroxidase-oxidase (PO) reaction is the haem-peroxidase catalysed reaction of molecular oxygen with NADH, and has been shown previously to behave in an oscillatory fashion. It has been used here as a useful laboratory model of the types of complex biochemical kinetics that are evinced by cellular systems. A laboratory apparatus for the study of this oscillator under conditions of steadystate substrate influx was designed and characterised. A standard set of experimental conditions was defined and a number of dynamical behaviours were observed using horseradish peroxidase; these included switching between coexistent steady-states (bistability), damped and sustained oscillations in the measurable components of the reaction when using NADH as the reducing agent. Special attention was paid to the methods of measurement of the mass-transfer of oxygen across the gas-liquid interface, and the determination of the rate constants of two models of the process. The reproducibility of the rate of oxygen mass-transport under standard conditions was also examined. A study of the physical and chemical parameters affecting the reaction was carried out. Through a series of experiments at different pH values and using two different buffers, it was found that the PO reaction has a pH optimum at approximately 5.0. Two pH indicators were tested for use with the oscillator: chlorophenol red (3',3"-dichlorophenolsulphonphthalein) and methyl red (4-dimethylaminobenzene-2'- carboxylic acid), and both were found to inhibit NADH oxidation. The effect of ionic strength was examined, and it was found that low ionic strengths favour oscillatory kinetics. The role of free radicals in the mechanism of the oscillator was probed by the use of perturbants such as superoxide dismutase and ascorbic acid, both of which were found to inhibit nonlinear oxidation of NADH. Tyramine (4-[2-aminoethyl]phenol), 4-aminophenol and 2,6-dichlorophenol were each tested for use in the PO reaction as in place of 2,4-dichlorophenol, the most commonly used promoter of oscillations. Tyramine was found to weakly stabilise oscillations, and its conversion to dityramine, a reaction shown to be catalysed by horseradish peroxidase, was taken to be indirect evidence for the formation of a phenolic free radical. 2,6-dichlorophenol was shown to be capable of generating sustained, and 4-aminophenol to completely inhibit, oscillations. Three mathematical models of the peroxidase-oxidase reaction were developed. Starting with a realistic model based on published and experimentally-verified rate constants which gave rise to similar oscillations to those observed in laboratory experiments, two successive reductions in the number of variables were made, generating two further models that like the first showed oscillations when integrated numerically on a computer. The simplest model, in two variables, views the basis of the peroxidase-oxidase reaction as being the interconversion of two forms of an enzyme acting upon a substrate intermediate, the assumed autocatalytic nature of the reaction providing the nonlinearity required for oscillations. A stability analysis of the model was carried out, revealing the presence of a Hopf bifurcation, a fact that was verified in computer simulations.