Kinetic Studies of Dehaloperoxidase-Hemoglobin From Amphitrite Ornata

Abstract

The focus of this research was to determine how three different factors affect the kinetics of the reaction between Dehaloperoxidase — Hemoglobin (DHP-Hb), a halogenated phenol substrate, and a peroxide co-substrate. The factors examined were the order of addition of the substrate and co-substrate, the pH of the solutions, and the difference between using hydrogen peroxide (H2O2) and m-Chloroperbenzoic acid (MCPBA) as the co-substrates. The order of addition studies were carried out on a stop-flow apparatus. The results presented here demonstrate that the substrate must bind to DHP-Hb prior to the addition of the peroxide co-substrate. The effect of pH was examined using stop-flow and photodiode array spectrometry. These studies show how the pKa of the substrate affects the mechanism of the reaction. The kinetic results actually depend on the pKa of the substrate used. Stop-flow studies and UV-Vis assays were used to compare H2O2 and MCPBA as co-substrates. These studies demonstrate that the use of MCPBA as the co-substrate yields a faster rate of product formation. The use of H2O2 as the co-substrate results in a larger amount of product formation at all pHs studied, with the exception of pH 6.0.