Awasthi Y C, Beutler E, Srivastava S K
J Biol Chem. 1975 Jul 10;250(13):5144-9.
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.
谷胱甘肽过氧化物酶已从人红细胞中纯化至同质。纯化步骤包括溶血产物的硫酸铵沉淀、CM - 纤维素（CM - 52）、DEAE - 纤维素（DE52）、葡聚糖凝胶G - 200和DEAE - 葡聚糖凝胶柱色谱。在最后一步，即DEAE - 葡聚糖凝胶A - 25柱色谱中，酶在一个主峰和拖尾部分被洗脱。在聚丙烯酰胺圆盘电泳上发现主峰是同质的，并被命名为谷胱甘肽过氧化物酶A（GSH - Px A）。然而，拖尾部分在聚丙烯酰胺圆盘电泳上分离成两条蛋白带。其中一条带对应于GSH - Px A，而另一条带迁移较慢，被命名为GSH - Px B。GSH - Px A和GSH - Px B每毫克蛋白的比活性分别为103和4酶单位。已发现针对同质GSH - Px A产生的抗体与GSH - Px B发生交叉反应。GSH - Px A和B都是硒蛋白。已发现GSH - Px A每摩尔蛋白含有3.5克原子硒。然而，由于材料不足，无法准确测定GSH - Px B的硒含量。通过沉降平衡法测定的GSH - Px A的分子量为95,000正负3,000。在尿素 - 十二烷基硫酸钠 - 聚丙烯酰胺圆盘电泳上，GSH - Px A和B解离成单个亚基。GSH - Px A亚基的分子量为23,000，GSH - Px B亚基的分子量为47,000。因此，GSH - Px A似乎是一种四聚体。我们的结果表明，GSH - Px B可能是主要成分GSH - Px A或其前体的一种改变形式。已对GSH - Px A的性质进行了研究。发现其等电点pH为4.9，酶活性的最适pH为8.5。活化能为8.2千卡。该酶对谷胱甘肽的Km为4.1 mM，而对叔丁基过氧化氢的Km为52μM。还研究了巯基试剂和金属离子对该酶的影响。
