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 designated 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 ± 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 μM. The effect of sulfhydryl reagents and the metal ions on the enzyme was also studied.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1975|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology