Coban T Abdulkadir, Senturk Murat, Ciftci Mehmet, Kufrevioglu O Irfan
Erzincan University, Department of Chemistry, Erzincan, Turkey.
Protein Pept Lett. 2007;14(10):1027-30. doi: 10.2174/092986607782541060.
In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.
在本研究中，我们研究了某些金属离子对人红细胞谷胱甘肽还原酶的抑制作用。为此，首先使用2',5'-ADP琼脂糖4B亲和凝胶和葡聚糖凝胶G-200凝胶过滤色谱法将人红细胞谷胱甘肽还原酶纯化了1051倍，产率为41%。进行SDS聚丙烯酰胺凝胶电泳以控制酶的纯化。SDS聚丙烯酰胺凝胶电泳显示酶为单一条带。在纯化过程中保持恒温（4℃）。使用分光光度计通过Beutler方法在340nm处测定酶活性。Hg(2+)、Cd(2+)、Pb(2+)、Cu(2+)、Fe(3+)和Al3+在体外对该酶表现出抑制作用。通过Lineweaver-Burk图以及绘制活性百分比与[I]的关系曲线来确定金属离子的K(i)常数和IC(50)值。Pb(2+)、Hg(2+)、Cu(2+)、Cd(2+)、Fe(3+)和Al(3+)的IC(50)值分别为0.011、0.020、0.0252、0.0373、0.209和0.229mM，K(i)常数分别为0.0254±0.0027、0.0378±0.0043、0.0409±0.0048、0.0558±0.0083、0.403±0.043和1.137±0.2mM。Pb(2+)、Hg(2+)、Cd(2+)和Fe(3+)表现出竞争性抑制，而其他离子则表现出非竞争性抑制。
