Purification and Properties of Human Erythrocyte Glutathione Peroxidase

Glutathione peroxidase has been purified to homogeneity from human erythrocytes. The purification steps involved ammonium sulfate precipitation of hemolysate, CM-cellulose (CM-52), DEAE-cellulose (DE52), Sephadex G-200, and DEAE-Sephadex column chromatography. In the last step, i.e. DEAE-Sephadex A-25 column chromatography, the enzyme was eluted in a major peak and tailing fraction. The major peak was found to be homogeneous on polyacrylamide disc electrophoresis and disignated as glutathione peroxidase A (GSH-Px A). The tail fraction, however, separated into two protein bands on polyacrylamide disc electrophoresis. One of the bands corresponded to GSH-Px A while the other band was slower moving and was designated as GSH-Px B. GSH-Px A and GSH-Px B had specific activity of 103 and 4 enzyme units per mg of protein, respectively. Antibodies raised against the homogeneous GSH-Px A have been found to cross-react with GSH-Px B. Both, GSH-Px A and B are selenoproteins. GSH-Px A has been found to contain 3.5 g atoms of selenium per mol of protein. Selenium content of GSH-Px B, however, could not be determined accurately due to insufficient material. The molecular weight of GSH-Px A as determined by the sedimentation equilibrium method is 95,000 plus or minus 3,000. On urea-sodium dodecyl sulfate-polyacrylamide disc electrophoresis GSH-Px A and B dissociate into single subunits. The molecular weight of the subunits of GSH-Px A is 23,000 and that of GSH-Px B is 47,000. Thus, it appears that GSH-Px A is a tetramer. Our results suggest that GSH-Px B is probably an altered form of the major component, GSH-Px A, or its precursor. The properties of GSH-Px A have been studied. The isoelectric pH was found to be 4.9 and the optimum pH for enzyme activity was 8.5. The energy of activation was 8.2 kcal. The Km of the enzyme for GSH was 4.1 mM while the Km for t-butyl hydroperoxide was 52 mu-M. The effect of sulfhydryl reagents and the metal ions on the enzyme was also studied.