Characterization of glutathione peroxidase in frog retina.

The properties and kinetic parameters of glutathione peroxidase were measured in retinal homogenates from frogs (R. pipiens) using a spectrophotometric assay in which the oxidation of glutathione is coupled to the oxidation of NADPH+ by exogenous glutathione reductase. The standard assay utilized 5.0 mM glutathione and 0.6 mM cumene hydroperoxide in 50 mM sodium phosphate buffer at pH 7.0. All solutions were bubbled with argon and all reactions were carried out under an atmosphere of argon. The enzyme activity was linear with protein concentration at different concentrations of both substrates. Determination of the pH optimum was complicated by a large increase in non-enzymatic oxidation of glutathione at alkaline pH. The highest ratio of enzymatic to non-enzymatic activity was at pH 7.0. Increasing glutathione concentration showed less effect on the spontaneous reaction than increasing the cumene hydroperoxide concentration. Glutathione peroxidase Km value for glutathione was 3.86 mM and for cumene hydroperoxide was 0.55 mM. Vmax for glutathione at 0.6 mM cumene hydroperoxide was 138 nmoles glutathione oxidized/min/mg protein, while at 5.0 mM glutathione the value for cumene hydroperoxide was 146 nmoles glutathione oxidized/min/mg protein. These studies demonstrate that glutathione peroxidase is active in the retina and establish the optimal experimental conditions for determination of the enzymatic activity.