Podzuweit H G, Schneider K, Knüttel H
Abteilung Immunologie der Universität Kiel, F.R.G.
Biochim Biophys Acta. 1987 Dec 11;905(2):435-46. doi: 10.1016/0005-2736(87)90473-1.
Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.
产碱杆菌H16的膜结合氢化酶是一种整合膜蛋白，只能通过去污剂处理使其溶解，而产碱杆菌模式菌株的膜结合氢化酶在细胞破碎后以可溶形式存在。对于产碱杆菌H16的这种酶，开发了一种新的高效纯化方法，包括用Triton X-114进行相分离以及在Procion Blue H-ERD-Sepharose上进行三嗪染料层析。纯化后得到了均一的氢化酶制剂，其比活性为269 U/mg蛋白（亚甲蓝还原），产率为45%。在纯化和储存过程中，0.2 mM MnCl2的存在能使该酶得到最佳稳定。产碱杆菌模式菌株的氢化酶通过类似方法从可溶提取物中纯化得到，但其比活性和活性产率较低。对这两种纯化酶的比较显示无显著差异：它们具有相同的分子量，均由两个不同亚基组成（Mr = 62,000、31,000），且等电点均接近pH 7.0。它们具有相同的电子受体特异性，与相似的高反应速率和相似的Km值反应。被还原的受体包括紫精染料、黄素、醌、细胞色素c、亚甲蓝、2,6 - 二氯酚靛酚、吩嗪硫酸甲酯和铁氰化物。泛醌和NAD未被还原。这两种氢化酶在免疫学上是相同的，且具有相同的电泳迁移率。对于产碱杆菌H16的膜结合氢化酶，已证明这种类型的氢化酶在其溶解、纯化状态下也能够催化逆反应，即从还原的甲基紫精产生H2。
