Seefeldt L C, Arp D J
Biochimie. 1986 Jan;68(1):25-34. doi: 10.1016/s0300-9084(86)81064-1.
Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.
维涅兰德固氮菌氢化酶已从细胞膜中纯化至同质。该酶先用 Triton X - 100 增溶，然后经硫酸铵 - 己烷萃取以去除脂质和去污剂。接着通过羧甲基 - 琼脂糖和辛基 - 琼脂糖柱色谱法对酶进行纯化。所有纯化步骤均在连二亚硫酸盐和二硫苏糖醇存在的厌氧条件下进行。该酶从细胞膜开始纯化了 143 倍，比活性达到 124 μmol H₂摄取·min⁻¹·mg 蛋白⁻¹。氢化酶的非变性聚丙烯酰胺凝胶电泳显示一条同时具有活性和蛋白染色的条带。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示两条分别对应于 67,000 和 31,000 道尔顿肽段的条带。SDS 凝胶的光密度扫描表明两条带的摩尔比为 1.07 ± 0.05。通过三种不同方法测定了天然酶的分子量。凝胶渗透法测得分子量为 53,000，蔗糖密度梯度离心法和天然聚丙烯酰胺凝胶电泳法分别测得分子量为 98,600 ± 10,000 和 98,600 ± 2,000。我们得出结论，维涅兰德固氮菌氢化酶是一种αβ二聚体（98,000 道尔顿），亚基分别为 67,000 和 31,000 道尔顿。镍和铁的分析表明，每摩尔氢化酶含 0.68 ± 0.01 摩尔镍和 6.6 ± 0.5 摩尔铁。该酶的等电点为 6.1 ± 0.01。此外，还研究了该酶的几种催化特性。H₂的 Km 值为 0.86 μM，并且在存在还原型甲基紫精的情况下观察到了 H₂的释放。已确定以亚甲蓝作为电子受体时酶活性的 pH 曲线，以及各种电子受体的 Km 和 Vmax。
