Suicide inactivation of peroxidase by H2O2: kinetic equations for peroxidatic oxidation reaction of guaiacol and determination of the kinetic parameters.

The kinetics of horseradish peroxidase (HRP) in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Those ranges of concentrations were selected in which: 1) the reaction was first-order in relation to [AH] and 2) [H2O2] >> [AH]. By combination of rate equations of the two concurrent reactions, consumption of AH and suicide inactivation of HRP, the overall kinetic equations were obtained which define the progress curve of reactions. The compatibility between equations and kinetic behaviour of reaction were evaluated in different ways. A close match was found between equations and experimental data. These equations can be used for determining 1) intact value of enzyme activity at start time of reaction and 2) apparent rate constant of suicide inactivation (ki') in a given concentration of H2O2, by processing of the data of the progress curve. The ki' value was found to be 0.178 +/- 0.003 min.-1 at 10 mM of H2O2 and 0.04 +/- 0.002 at 3 mM H2O2, at 27 degrees C and sodium phosphate buffer, pH = 7.0.