Stults L W, Moshiri F, Maier R J
J Bacteriol. 1986 Jun;166(3):795-800. doi: 10.1128/jb.166.3.795-800.1986.
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.
我们通过亲和层析从自由生活的日本根瘤菌中纯化出了活性氢化酶。日本根瘤菌的摄取氢化酶此前一直被视为一种对氧敏感的蛋白质。然而，在此次纯化过程中，既未添加还原剂，也未尝试排除氧气。事实上，向需氧纯化的蛋白质中添加连二亚硫酸钠会导致活性迅速丧失。纯化后的氢化酶在氧气环境下储存比在氩气环境下储存更稳定。用氯化钠洗涤过的氢氧化氢气膜在 Triton X - 100 和脱氧胆酸盐中溶解，然后加载到活性红 120 - 琼脂糖柱上。纯化后的氢化酶在 0.36 M 氯化钠浓度下洗脱，含有镍，最适 pH 为 6.0。纯化倍数达到 452 倍，比活性为每毫克蛋白质每分钟氧化 76.9 μmol 的氢气，产率为 17%。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示其亚基的估计分子量分别为 65,000 和 33,000。以这种方式制备的氢化酶被用于产生针对这两个亚基的抗体并进行亲和纯化。
