Carbon monoxide oxygenase activity of cytochrome cd1.
Carbon monoxide oxygenase activity of cytochrome cd1.
复制标题
细胞色素 cd1 的一氧化碳加氧酶活性。
DOI：
10.1021/bi00414a064
复制
发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Thrasher,JS
中科院分区：
文献类型：
--
作者：
Timkovich,R;Thrasher,JS
材料和方法培养铜绿假单胞菌（ATCC 19428）并如前所述（Timkovich和Cork，1982）纯化细胞色素。通过加入固体硫酸铵至90%饱和度使其沉淀，离心后回收沉淀。将蛋白质重新溶解于含有5mM EDTA的0.1M磷酸钾缓冲液（pH 7.0）中，所述缓冲液已通过Nalgene灭菌过滤器单元灭菌。将0.5 mL单个样品在液氮中冷冻备用。用标准消光系数测定蛋白质浓度（Silvestrini等人，1979年），并将报告为亚基的浓度。13 CO，99.4原子% 13 C，购自MSD Isotopes。其他研究纯度的气体购自Matheson。构建了特殊的细胞来测量水酶溶液上方的气相的红外光谱。采用了两种类型，不同之处仅在于对外部大气的密封类型。第一种是将内径为1cm、长2.5cm的导管与内径为2.3cm、外径为2.5cm、长10cm的导管以直角融合。较小的侧管配有标准橡胶隔膜，气体和试剂可以通过针头添加。第二种类型旨在确保更高的密封完整性。小边管
Materials and MethodsPseudomonas aeruginosa (ATCC 19428) was cultured and cyt cd\purified as described previously (Timkovich & Cork, 1982). It was precipitated by the addition of solid ammonium sulfate to 90% saturation, and the pellet was recovered after centrifugation. Protein was redissolved in 0.1 M potassium phosphate buffer with 5 mM EDTA, pH 7.0, that had been sterilized by passage through a Nalgene sterilizing filter unit. Individual samples of 0.5 mL were frozen in liquid nitrogen until use. Protein concentration was determined with standard extinction coefficients (Silvestrini et al., 1979) and will be reported as the concentration of subunits. 13CO, 99.4 atom% 13C, was purchased from MSD Isotopes. Other gases of research purity were purchased from Matheson. Special cells were constructed to measure the infrared spectrum of the gas phase above an aqueous enzyme solution. Two types were employed that differed only in the type of seal against the outside atmosphere. In the first type a tube 2.5 cm long with 1-cm id was fused at a right angle to a tube 10 cm long with 2.3-cm id and 2.5-cm od. The smaller side tube was fit with a standard rubber septum where gases and reagents could be addedby a needle. The second type was designed to ensure a higher integrity seal. The small side tube