Aerobic purification of hydrogenase from Rhizobium japonicum by affinity chromatography-猫眼课题宝

亲和层析有氧纯化日本根瘤菌氢化酶

DOI：

发表时间：

1986

期刊：

Journal of Bacteriology

影响因子：

3.2

通讯作者：

中科院分区：

生物学3区

文献类型：

作者：

L. Stults;F. Moshiri;R. Maier

采用亲和层析法从日本根瘤菌中分离纯化了活性氢化酶。R.日本血吸虫以前被认为是一种氧敏感蛋白。然而，在该纯化中，不添加还原剂，也不试图排除氧。事实上，向需氧纯化的蛋白质中加入连二亚硫酸钠导致活性迅速丧失。纯化的氢化酶在O2下储存比在Ar下储存更稳定。将氯化钠洗涤的氢氧化膜溶解在Triton X-100和脱氧胆酸盐中，并加载到活性红120-琼脂糖柱上。纯化的氢化酶在0.36 M NaCl下洗脱，含有镍，最适pH为6.0。有452倍的纯化，导致在一个特定的活性为76.9 mumol的H2氧化每分钟每毫克蛋白质和产率为17%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示亚基，估计分子量为65，000和33，000。以这种方式制备的氢化酶用于产生和亲和纯化针对两种亚基的抗体。

We purified active hydrogenase from free-living Rhizobium japonicum by affinity chromatography. The uptake hydrogenase of R. japonicum has been treated previously as an oxygen-sensitive protein. In this purification, however, reducing agents were not added nor was there any attempt to exclude oxygen. In fact, the addition of sodium dithionite to aerobically purified protein resulted in the rapid loss of activity. Purified hydrogenase was more stable when stored under O2 than when stored under Ar. Sodium-chloride-washed hydrogen-oxidizing membranes were solubilized in Triton X-100 and deoxycholate and loaded onto a reactive red 120-agarose column. Purified hydrogenase elutes at 0.36 M NaCl, contains a nickel, and has a pH optimum of 6.0. There was 452-fold purification resulting in a specific activity of 76.9 mumol of H2 oxidized per min per mg of protein and a yield of 17%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed subunits with estimated molecular weights of 65,000 and 33,000. Hydrogenase prepared in this manner was used to raise and affinity purify antibodies against both subunits.

Desulfovibrio vulgaris hydrogenase: a nonheme iron enzyme lacking nickel that exhibits anomalous EPR and Mössbauer spectra.