Harker A R, Xu L S, Hanus F J, Evans H J
J Bacteriol. 1984 Sep;159(3):850-6. doi: 10.1128/jb.159.3.850-856.1984.
The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.
通过化学无机营养方式生长的日本根瘤菌的摄取氢化酶被纯化至表观均一，最终比活性为每分钟每毫克蛋白质氧化69微摩尔氢气。该纯化过程包括用Triton提取破碎的细胞膜以及进行DEAE - 纤维素和Sephacryl S - 200柱色谱。纯化后的蛋白质含有两条多肽链，仅通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳才能分离。它们在非变性聚丙烯酰胺凝胶和蔗糖密度梯度中共同迁移。分子量约为60,000和30,000。十二烷基硫酸钠凝胶的光密度扫描表明摩尔比为1.03±0.03。制备了针对60千道尔顿多肽的抗血清，用于通过酶联免疫吸附测定法检测氢化酶。该抗血清与30千道尔顿多肽无交叉反应。在含有63Ni的条件下生长的经Triton提取的细胞的非变性凝胶电泳显示氢化酶与放射性Ni共同迁移。
