Oxidation states of glutathione peroxidase

This chapter discusses a method that has been developed for the reproducible isolation of different oxidized forms of glutathione peroxidase, which can be differentiated by their relative stability and by their reactivity with cyanide. The procedure is convenient for isolating substantial amounts of GSH-peroxidase from relatively small volumes of blood (1-2 liters), using conventional isolation procedures. The removal of hemoglobin is accomplished without the use of organic solvents. The sequence of operations is designed to eliminate the need for the concentration of the enzyme by ultrafiltration, a procedure that may cause extensive loss of activity. In the final stages of chromatography, the inclusion of 10% ethanol stabilizes the enzyme when it is in an oxidized form. The use of a continuous-flow rotor in the ammonium sulfate fractionation step has made it possible to obtain highly active enzyme with yields in the range of 60% or more. A second Sephadex G-150 chromatography following the hydroxyapatite chromatography gives a slightly purer form of enzyme. The ratio of enzyme activity to Se content attains a maximum early in the purification and then remains constant. This ratio provides a means of detecting the conversion of the enzyme to a form containing selenium in nonfunctional forms in the course of purification.