Kinetics of hypothiocyanite production during peroxidase-catalyzed oxidation of thiocyanate

Abstract

We report here a kinetic study of the generation of hypothiocyanite (OSCN −), a product of lactoperoxidase-catalyzed oxidation of thiocyanate ion. Previous studies have measured OSCN − by reactions involving the oxidation of sulfhydryl compounds. Our results show that a more suitable kinetic analysis of OSCN − can be based on absorbance changes measured at 235 nm. About 90% of the oxidation products of SCN − observed at 235 nm were reactive with sulfhydryls and could be reduced with 2-mercaptoethanol. Both thiocyanate and peroxide were rate-limiting and the formation of OSCN − was proportional to the initial concentration of H 20 2 until an equimolar concentration of H 20 2 and SCN − was reached. This equimolar concentration gave the maximum generation of OSCN −. High concentrations (> 100 μg/ml) of lactoperoxidase decreased OSCN − generation, but only if the SCN − was added to the enzyme prior to addition of H 20 2. With lactoperoxidase concentrations exceeding 1 μg/ml, the reaction velocity was rapid, but the decay of OSCN − was slow. Free H 20 2 in the reaction mixture always resulted in rapid decay of OSCN −. Addition of varying concentrations of peroxide to solutions containing 1 μg/ml of enzyme and [SCN −]=5 mM gave a family of hyperbolic A 235 vs. time curves. Both the initial slopes and the plateaus of these curves increased linearly with increasing initial peroxide concentrations up to [H 20 2]=0.4 mM, remained relatively constant in the range [H 20 2]=0.4 to 0.8 mM, and decreased rapidly above [H 20 2] =0.8 mM. These resuits are consistent with the following kinetic model: Hydrogen peroxide reacts rapidly with lactoperoxidase to produce compound I. This compound I oxidizes SCN − to OSCN − and also oxidizes OSCN − to O 2SCN −. The OSCN − also reacts with SCN −. The formation of OSCN − is associated with the appearance of an absorbance peak at 225–235 nm. The oxidation of OSCN − by excess peroxide or its decomposition at high concentrations is associated with a decrease in A 235 and the appearance of a peak at 245–255 nm. The extinction coefficient for OSCN − was determined to be 1.29·10 3 M −1·cm −1. The second-order rate constant for the oxidation of thiocyanate by compound I was estimated to be 2.10 5 M −1· −1.